is or · however, that 5mt is incorporated by tryptophan-starved e. coli (lark 1970; mosteller and...

REPRESSOR AND ANTIREPRESSOR IN THE REGULATION OF STAPHYLOCOCCAL PENICILLINASE SYNTHESIS

JOHN IMSANDE

Departments of Genetics and Biochemistry, Iowa State University, Ames, Iowa 50010

Manuscript received March 26, 1973

ABSTRACT

5-methyltryptophan (5MT) induces penicillinase synthesis in Staphylococ- cus aureus. The analog is incorporated into protein by both wild-type and tryptophan-starved cells. Since normal penicillinase repressor appears to contain tryptophan even though penicillinase itself does not, it is concluded that 5MT induces penicillinase synthesis by becoming incorporated into the penicillinase repressor and thereby inactivating the repressor. Thus biochemical data support the existence of a penicillinase repressor and indicate that penicillinase synthesis is regulated by negative control and not by positive control.-In the absence of exogenous tryptophan, staphylococcal penicillinase induction can be inhibited by 7-azatryptophan (7azaT). Because 7azaT is incorporated into protein by tryptophan-starved cells, it is concluded that 7azaT blocks penicillinase induction by inactivating a penicillinase regulatory protein into which the analog has been incorporated. Incorporation of 7azaT does not appear to inactivate the operator binding site or the effector binding site on the penicillinase repressor. Therefore, it appears that 7azaT blocks penicillinase induction by inactivating the penicillinase antirepressor, a protein required for inactivation of the penicillinase repressor and, hence, required for penicillinase induction.

N a previous report IMSANDE, ZYSKIND and MILE (1972) showed that several I tryptophan analogs induce penicillinase synthesis in at least two strains of Staphylococcus aureus and that the 5-methyltryptophan-induced synthesis of penicillinase is rapidly halted when tryptophan is added to the culture medium. They postulated that 5-methyltryptophan (5MT) might induce penicillinase synthesis either by becoming incorporated into cellular proteins, including the penicillinase regulatory proteins, and thereby producing inactive repressor, or by serving as a structural analog of the natural inducer, penicillin. Also, they pro- vided evidence that during penicillinase induction the penicillinase repressor is inactivated by a genetically specified antirepressor protein rather than by the inducer, or effector, per se.

In an attempt to determine the mechanism, or mechanisms, whereby 5MT induces penicillinase synthesis in S. aureus and to gain greater insight into the regulation of penicillinase synthesis in general, it was necessary to establish whether or not SMT is incorporated into protein. PARDEE and PRESTIDGE (1958)

IThis investigation was supported In part by a grant from the Iowa Division of the American Cancer Society. This report is Journal Paper J-7421 of the Iowa Agriculture and Home Economics Experiment Station, Ames (Project No. 1821).

Genetics 75: 1-17 September, 1973.

2 J. IMSANDE

reported that 5MT stimulates protein synthesis in tryptophan-starved E . coli. Subsequently, it was reported that 5MT could not be incorporated (MUNIER and COHEN 1959) nor could i t be attached to tRNA by the tryptophanyl-tRNA synthetase of E. coli (DOOLITTLE and YANOFSKY 1968). More recent data indicate, however, that 5MT is incorporated by tryptophan-starved E. coli (LARK 1970; MOSTELLER and YANOFSKY 1971) and B. subtilus (BARLATI and CIFERRI 1970). Similar evidence indicates that 7-azatryptophan is also incorporated into protein by E. coli (PARDEE and PRESTIDGE 1958; MOSTELLER and YANOFSKY 1971).

We wish to report that 4MT, 5MT, 6MT, and 7-azatryptophan all stimulate amino acid incorporation in tryptophan-starved S. aureus; however, under these conditions, only 5MT and 6MT are efficient inducers of penicillinase synthesis. On the other hand, addition of 5MT to the culture medium inhibits amino acid incorporation approximately 30%. Thus, although 5MT reduces the overall rate of protein synthesis in the wild-type cell, it is nevertheless incorporated into staphylococcal proteins and thereby induces penicillinase synthesis.

MATERIALS A N D METHODS

Organisms and media: All bacterial strains, media, and culture conditions used in this study have been described (IMSANDE, ZYSKIND and MILE 1972; ZYSKIND and IMSANDE 1972). The bacterial inoculum for each experiment was obtained from a fresh (18-hr) slant fortified with either Brain Heart Infusion medium or casamino acid medium as described. All slants contained 0.25 mM Cd(NO,),. When synthetic medium was used, trace minerals were added (NOVICK 1963). All media were warmed before the addition of the cells.

Assays: Penicillinase activity was assayed by the standard procedure (PERRET 1954). Cell growth was followed with a Zeiss spectrophotometer at a wavelength of 535 nm. Amino acid analysis was performed by the procedure of LIU and CHANG (1971). Incorporation of radioactivity into protein was determined by the following procedure: individual culture samples were transferred into an equal volume of cold 10% trichloroacetic acid, and after 30 min on ice, the samples were heated at 90" for 20 min, collected on a filter membrane, and washed with 5% trichloroacetic acid. The filter membranes were dried at 90" for 1 hr; toluene-based scintillation fluid was added; and radioactivity was measured with a Packard scintillation spectrometer.

Materials: 4-methyltryptophan (4MT), 5-methyltryptophan (5MT), 6-methyltryptophan (6MT), 7-azatryptophan (7azaT), tryptophan (Trp) , 5-fluorotryptophan (5FT), tryptamine, and 5-methyltryptamine (5M tryptamine) were obtained from Sigma Chemical Co., St. Louis, MO.; L-tryptophan-3-C14 (side chain labeled) and 3H L-isoleucine were products of New Eng- land Nuclear, Boston, Massachusetts. 2(2'-carboxyphenyl) benzoyl-6-amino-penicillianic acid (CBAP) was a gift of Imperial Chemical Industries, Ltd., Macclesfield, England.

RESULTS

Zncorporation of 5-methyltryptophan: Certain analogs of tryptophan are known to induce penicillinase synthesis in S. aureus (IMSANDE, ZYSKIND and MILE 1972). In an attempt to determine whether the analogs might induce penicillinase synthesis by blocking the formation of repressor o r by causing the formation of faulty repressor, a tryptophan auxotroph was deprived of tryptophan and penicillinase activity was assayed. As shown in Table 1 (lines 1 and 2), the basal level of staphylococcal penicillinase increased only slightly during tryptophan starvation. Hence blocking overall protein synthesis by depriving the cell of an

REGULATION O F PENICILLINASE SYNTHESIS

TABLE 1

Effect of tryptophan starvation and tryptophan analogs during tryptophan starvation on the induction of staphylococcal penicillinase

3

Addition' Penicillinase Cell mass Specific (units/ml) (mg dry wt/mU activity

None 0.73 0.092 7.9 trp(5 pg/ml) 0.79 0.137 5.0 4MT ( 100 pg/ml) 0.93 0.107 8.7 5MT(100 pg/ml) 4.67 0.110 42.5 6MT(100 pg/ml) 4.09 0.104 39.3 7azaT (1 00 pg/ml) 0.79 0.102 7.7 5MT ( 100 pg/ml) + Trp ( 5 pg/ml) 0.72 0.140 5.1

* Cells [655 t ~ - ( P 1 ~ ~ ~ ) ] were g r o w n into log phase on CAA medium + trp (201gg/ml), collected on a filter membrane, washed, suspended in synthetic medium lacking tryptophan at a cell mass of 0.05 mg dry weight/ml. Additions were made as indicated above, the cultures were grown at 37" for 90 min, and penicillinase activity and cell density were determined.

amino acid not present in penicillinase (AMBLER ar,d MEADWAY 1969) does not induce penicillinase synthesis appreciably. Addition of 5MT or 6MT to tryptophan-starved cells, however, produced an eightfold increase in enzyme (lines 4 and 5 ) , but addition of 4MT or 7azaT did not induce penicillinase synthesis significantly (compare line 1 with lines 3 and 6). The data in column 3 suggest that all of the analogs tested supported an increase in cell mass. Thus 5MT and 6MT do not induce penicillinase simply by blocking repressor synthesis. Instead, they stimulate protein synthesis in tryptophan-starved cells and, hence, seem to be incorporated into protein.

Therefore, the extent and relative effectiveness of tryptophan analogs in the stimulation of protein synthesis during tryptophan starvation was examined (Figure 1). These data show that the commercial preparations of all of the analogs tested stimulate protein synthesis in tryptophan-starved cells. If these preparations are free of tryptophan, then the analogs would certainly seem to be incorporated into protein. An amino acid analysis of approximately 0.1 6 @moles of the tryptophan or 5MT used in these studies was performed in the presence of thioglycolic acid by the procedure of LIU and CHANG (1971). Although the elution of 5MT was accompanied by an impurity, it nevertheless was found to be free of tryptophan (unpublished results). Hence 5MT must be incorporated into protein by S. aureus. Direct verification of this contention was initially im- possible because histidine and 5MT elute as one peak under the standard chro- matographic conditions employed (unpublished results). However, tryptophan is known to produce a characteristic fluorescence emission spectrum when ex- cited by near ultraviolet light. Therefore 5MT was examined for fluorescence and found to produce an emission spectrum very similar to that produced by tryptophan (TMSANDE, to be published). Hence a IGW concentration of 5MT in the presence of histidine can be identified and quantified by fluorescence analysis. Strain 655N (PI258) was therefore grown for two doublings on CAA medium supplemented with 100 pg/ml 5MT and protein was isolated from the washed

4 J. IMSANDE

15

aJ C

0

U

5 15 25 M i n u t e s

FIGURE 1 .-Relative effectiveness of tryptophan analogs in the stimulation of protein synthesis during tryptophan starvation. Cells [655 trp- (PI268)] were transferred from a fresh slant fortified with casamino acid (CAA) medium supplemented with tryptophan to CAA medium plus tryptophan. The cells were grown for 100 min, harvested, suspended in synthetic medium and grown for 50 min. Filtered cells were washed, resuspended in synthetic medium free of tryptophan and with the isoleucine content reduced to 5 pg/ml. The cells were tryptophan-starved for 40 min after which the culture was divided to 7 equal fractions. Each subculture (total volume 6.30 ml) was supplemented with 1.25 pCi 3H isoleucine and the indicated tryptophan derivative: 0, no supplement; hexagon, 5 @g/ml trp; A, 166 pg/ml4MT; U, 166 pg/ml5MT; A, 166 pg/ml6MT; e, 166 pg/ml 7 azatryptophan; W, 166 pg/ml5MT plus 5 pg/ml trp. The subcultures were shaken at 37", and 2 ml samples were removed from each flask at 5, 15 and 25 min after the addition of the indicated supplements.

cells by the procedure of BARLATI and CIFERRI (1970). The dried protein was hydrolyzed and 0.47 mg was subjected to chromatography as described by LIU and CHANG (1971) .The tryptophan peak and the combined histidine-5MT peak were collected for fluorescence analysis instead of being assayed with ninhydrin. The peaks contained 5.5n moles of tryptophan and 10.4 n moles of SMT, respec- tively. Thus, assuming complete recovery of both compounds, approximately 1.5 n moles of tryptophan and 10.4 n moles of 5MT were incorporated into the protein sample used for this determination during growth in the presence of 100 pgJml 5MT.

Inhibition of I4C tryptophan incorporation by 5MT also suggests that 5MT is incorporated into proteins. As shown in Figure 2, the extent of inhibition of 14C tryptophan incorporation by 5MT is dependent upon the concentration of exogenous tryptophan. These results suggest that 5MT competes with tryptophan

REGULATION O F PENICILLINASE SYNTHESIS 5

I 1 I I I

0 10 20 30 40 50 60

M i n u t e s

FIGURE %--Inhibition of 14C tryptophan incorporation by 5MT. Cells [655N(PI,,,)] were transferred from a fresh casamino acid-supplemented slant to CAA medium. After 120 min of growth, the cells were collected on a filter membrane, washed, suspended in CAA medium, and grown for an additional 20 min. The culture was diveded into 6 equal fractions, supplemented as indicated, and diluted to an optical density at 535 nm of 0.23: 0, 0.25 pg/ml 14C tryptophan; 0 , 0.25 pg/ml 14C trp plus 100 pg/ml 5MT; A, 0.50 pg/ml 14C trp; A, 0.50 pg/ml 1% trp plus 100 pg/ml5MT; 0, 1.0 pg/ml14C trp; W, 1.0 pg/ml 14C trp plus 100 pg/ml 5MT. For all cultures the specific activity of 14C tryptophan was 0.637. Samples (2 ml) were removed from each flask at the indicated times.

either at the transport level or at the amino acid activation step, or both. More significantly, however, there is a direct correlation between the extent of inhibition of 14C tryptophan incorporation by 5MT (Figure 2) and the effectiveness of 5MT as an inducer of penicillinase (Figure 3) .

Finally, if 5MT were to induce penicillinase synthesis as a free metabolite as was originally speculated (IMSANDE, ZYSKIND and MILE 1972) then one might expect 5-methyltryptamine to also induce penicillinase synthesis. All attempts to elicit a significant penicillinase induction with amino acid derivatives incap- able of becoming incorporated into protein were unsuccessful (Table 2 lines 5 and 6) .

6 J. IMSANDE

T u r b i d i t y (535 nm)

FIGURE 3.-Effect of low levels of tryptophan on the induction of penicillinase by 5MT. Cells [655N(PI,,,)] were grown in the media described in Figure 1, except exogenous tryptophan was not added nor were the cells tryptophan-starved. The culture was divided into 3 equal fractions, which were supplemented as follows: 0, 100 pg/ml5MT; hexagon, 100 pg/ml5MT plus 0.25 &g/ml trp; A, 100 pg/ml5MT plus 0.50 pg/ml trp. Samples (3 ml) were removed at zero- time and at IO-min intervals thereafter.

TABLE 2

Effect of tryptophan derivatives on penicillinase induction

Addition’

None 0.60 0.193 3.1 5MT 6.27 0.1G 44. 6MT 1.26 0.180 7.0 5FT 1.04 0.1 76 5.9 5MTTryptamine 0.63 0.173 3.6 Tryptamine 0.50 0.190 2.6 CBAP 16.5 0.195 84.

* Log phase cells [655N(PI,,,)] growing on CAA medium where divided into six subcultures and 100 pg/ml (final concentration) of the indicated compound was added to the individual flasks (except CBAP, which was at 7.25 pM).


TABLE 3

Effect of tryptophan derivatives on P-galactosidase synthesis

7

Additions' P-galactosidase activity Cell mass

(pmoles hydrolyzed/min/ml) (mg dry weight/ml)

None Gal-6-P Gal-6-P ,+ 5MT Gal-6-P + 7azaT

<0.002 0.032

<0.002 <0.002

1.13 1.35 0.85 0.91

* Strain 655N(PI,,,) was grown into log phase on CAA medium that contained P-glycerolphosphate as a carbon source instead of glucose. The cells were collected on a filter membrane, washed, and suspended in fresh CAA-P-glycerolphosphate medium at a cell density of 0.36 mg dry weight/ml. The cell suspension was divided into four equal fractions and 1 P M galactose-6- phosphate, the inducer of P-galactosidase, was added to three cultures. Also, 100 p g / d 5MT o r 7azatryptophan was added where indicated. After 3 hrs of growth, cell density and P-galactosidase activity (MORSE et al. 1968) were determined.

Therefore it is concluded that 5MT induces penicillinase synthesis in S. aureus by becoming incorporated into protein and thereby causing the formation of faulty penicillinase repressor. The conclusion that 5MT is incorporated into staphylococcal protein is supported by the fact that growth in the presence of 5MT or 7-azatryptophan blocks the induction and, or, synthesis of staphylococcal /3-galactosidase (Table 3 ) .

Deinduction during tryptophan deprivation: Staphylococcal penicillinase is known to lack tryptophan (AMBLER and MEADWAY 1969). If the penicillinase repressor contains tryptophan, as the data presented above indicate, then the rate of penicillinase deinduction should be dependent upon the availability of tryptophan to the cell. To test this prediction a tryptophan auxotroph was induced by CBAP in the presence of tryptophan and subsequently grown in the absence of both CBAP and tryptophan (Figure 4). The data show that the addition of tryptophan to the culture medium during deinduction increases the rate of penicillinase deinduction, as would be expected if the repressor contains tryptophan.

Requirement of protein synthesis for penicillinase induction: Since penicillinase synthesis occurs when previously induced cells are deprived of tryptophan (Figure 4) one might expect that it would be possible to induce penicillinase formation with CBAP during tryptophan deprivation. However, as shown in Figure 5, tryptophan starvation prevents penicillinase induction. Hence, the synthesis of a tryptophan-containing protein seems to be an obligatory prerequisite for penicillinase induction.

Prevention of penicillinase induction by 7-azatryptophan: In the absence of exogenous tryptophan, 7-azatryptophan is incorporated into protein by S. aureus (Tables 1 and 3 and Figure 1). Nevertheless this analog does not induce penicillinase synthesis appreciably (Table 1 ) . Therefore, since synthesis of a tryptophan-containing protein is required for penicillinase induction (see Figure 5) ,

8 J. IMSANDE

0.25 0.30 0.35 0.40 0.45 0.50 0.55

T u r b i d i t y (535 nm)

FIGURE 4.-Kinetics of penicillinase deinduction during tryptophan starvation. Cells [655 trp- (PI258)] were grown on CAA medium, supplemented with tryptophan, as described in Figure 2. The culture was induced with 7.25 pM CBAP in the presence of ferrous ammonium sulfate for 60 min. The cells were collected on a filter membrane, washed, and suspended in CAA medium containing ferrous ions. Samples (3 ml) were removed for penicillinase assay at zero- time and at 4-min intervals thereafter, 0; at 41 min a subculture was started and 20 pg/ml trp was added to it immediately. Samples (3 ml) were als3 removed for penicillinase assay at 4 min intervals from the subculture (hexagon). The dashed line represents a separate experiment in which tryptophan was added immediately after removing the exogenous CBAP instead of 41 min later.

the ability of 7-azatryptophan to inhibit penicillinase induction was examined (Figure 6). It can be seen that 7-azatryptophan effectively prevents penicillinase induction by the classical penicillinase inducer CBAP. Data presented in Figure 7 show that the rate of penicillinase deinduction in the presence or absence of 7-azatryptophan is essentially identical. Therefore, growth in the presence of 7-azatryptophan does not uniquely prevent penicillinase synthesis. Also, in the presence of 20 ,pg/ml tryptophan, 7-azatryptophan does not inhibit penicillinase induction (Figure 8). Is it possible that 7-azatryptophan, in the absence of exogenous tryptophan, inhibits penicillinase induction by becoming incorporated into the tryptophan-containing protein described above?

Role of 5 M T and 7-azatryptophan in the regulation of penicillinase synthesis: According to the working model proposed previously, the regulation of penicillinase synthesis is controlled by both a repressor and antirepressor protein ( IM- SANDE, ZYSKIND and MILE 1972). Is it possible that the unidentified tryptophan-


M i n u t e s

FIGURE 5.-Blockage of penicillinase induction by tryptophan deprivation. Strain 655 trp- (PI& was grown into exponential phase on nutrient broth medium, collected on a membrane filter, washed and suspended in casamino acid medium supplemented with 20 pg/ml tryptophan. After 60 minutes’ growth in casamino acid medium, the cells were collected and suspended in synthetic amino acid medium lacking tryptophan and allowed to grow for an additional 20 minutes. CBAP (7.25 pM) was added and the culture was immediately divided into two equal fractions. One subculture was supplemented with 20 fig/ml tryptophan ( 0 - 0 ) and the other subculture received no supplement (0-0). Samples (3 ml) were removed from each culture for penicillinase assay immediately and at 5-minute intervals thereafter.

containing protein described above and the proposed antirepressor are equiva- lent? In an attempt to examine the validity of this proposition, cells were induced by 5MT in the presence or absence of 7-azatryptophan (Figure 9). It will be noted that 7-azatryptophan inhibits induction by CBAP more effectively than it inhibits induction by 5MT. These results are consistent with the notion that incorporation of 5MT causes the formation of faulty repressor while incorporation of 7-azatryptophan causes inactivation of the active antirepressor that is normally produced in the presence of CBAP. Also, the kinetics of penicillinase deinduction were compared in cultures induced by 5h4T and CBAP or by CBAP alone. It will be recalled that a CBAP-induced culture deinduces very slowly upon removal of exogenous inducer (Figure 4), although a 5MT-induced culture deinduces very rapidly when 5MT is removed or when tryptophan is added to the culture medium (IMSANDE, ZYSKIND and MILE 1972). As shown in Figure 10, the rate of deinduction of a CBAP-induced culture is dependent upon the length

10 J. IMSANDE

I I I I

0.40 0'

0 . 2 0 0.30

Turbidity (535nm)

FIGURE 6.-Inhibition of penicillinase induction by 7-azatryptophan. S. aurew strain 8325 (PI,?,) was grown into exponential phase on nutrient broth medium, collected on a membrane filter, washed, and suspended in casamino acid medium. After 60 minutes' growth in casamino acid medium, the cells were collected and suspended in fresh cnsamino acid medium and allowed to grow for an additional 20 minutes. The culture was divided into four equal fractions, and two subcultures were supplemented with 100 pg/ml 7-azatryptophan. Five minutes after the addition of 7-azatryptophan the inducer, CBAP, was added to one flask containing and one flask lacking 7-azatryptophan. 0, no addition; ., 7-azatryptophan; A, 7.25 pM CBAP; A, 7-azatryptophan + 7.25 pM CBAP. Samples (3 ml) were removed from each culture for penicillinase assay immediately after the addition of the penicillinase inducer, CBAP, and at 10-minute intervals thereafter.

of time that the cells had been in contact with the exogenous inducer (i.e., the longer the cells grew in the presence of the inducer the longer the deinduction period). Conversely, cells grown for 15 min in the presence of CBAP, followed by 15 or 30 min in the presence of both 5MT and CBAP, deinduced more rapidly than cells grown 15 min in the presence of CBAP alone (Figure 11). This ob- servation and a comparison of the rates of deinduction after 30 or 45 min contact with the inducers (Figs. 10 and 11) suggest that cell growth in the presence of 5MT causes a destruction of the material that is responsible for the relatively slow deinduction of penicillinase synthesis. These results (Figures 10 and 11) are consistent with the notion (IMSANDE, ZYSKIND and MILE 1972) that cell growth in the presence of SBAP causes active penicillinase antirepressor to accumulate within the cell, while growth in the presence of 5MT results in the synthesis of both inactive penicillinase repressor and inactive antirepressor.


Turbidity (535nm)

FIGURE 7.-Kinetics of penicillinase deinduction in the presence of 7-azatryptophan. S . uureus strain 8325 (PI524) was grown as described in Figure 6; 20 minutes after the second resuspension, however, the entire culture was induced with 7.25 p M CBAP in the presence of ferrous ions. After 60 minutes' growth in the presence of the inducer, the cells were collected, washed, and suspended in fresh casamino acid medium supplemented with ferrous ion, and the culture was divided. One subculture was diluted with fresh medium (0), and the other (X) was diluted with medium containing 7-azatryptophan (100 pg/ml final concentration). Samples ( 3 ml) were removed from each culture for penicillinase assay immediately after dilution and at 5-minute intervals thereafter for 50 minutes.

I I I A '

6 - - E

ul d C

2 5 -

- 4 - - - .- 0

C Q)

.- 3

a

-

- c

T u r b i d i t y ( 5 3 5 nm)

FIGURE 8.-Prevention of the 7-azatryptophan-dependent inhibition of penicillinase induction by tryptophan S. uureus strain 8325(PI,,,) was grown as described in Figure 6, and 20 pg/ml tryptophan was added to the culture 20 minutes after the resuspension. The culture was then divided into two fractions, and 7-azatryptophan (100 pg/ml final concentration) was added to one subculture (A), and the other subculture was not supplemented ( A ) . Five minutes after the addition of 7-azatryptophar1, both cultures were induced with 7.25 &M CBAP. Samples were removed for penicillinase assay immediately after the addition of CBAP and at 4-minute intervals thereafter.

12 J. IMSANDE

Turbidity (535 nm)

FIGURE g.--Induction of penicillinase synthesis by 5MT is not blocked by 7-azatryptophan. S. aureus strain 8325(PIs,,) was grown into exponential phase on nutrient broth medium, collected, grown in casamino acid medium, collected and grown for an additional 20 min on casamino acid medium as described in Figure 6. The culture was divided into four equal fractions and two subcultures were supplemented with 100 pg/ml 7-azatryptophan. Five minutes after the addition of 7-azatryptophan the cells were induced with CBAP or 5MT. 0, 7.25 pM CBAP; 0 , 7.25 pM CBAP $. 7-azatryptophan; A, 100 pg/ml5MT; A, 100 pg/ml5MT + 7-azatryptophan. Samples (3 ml) were removed from each culture for penicillinase assay immediately and et 10-minute intervals thereafter.

DISCUSSION

The penicillinase produced by S. aureus lacks tryptophan (AMBLER and MEAD- WAY 1969). Thus, in theory, a tryptophan auxotroph can synthesize penicillinase de novo during tryptophan deprivation. Since, however, the penicillinase produced by S. aureus is an inducible enzyme whose synthesis is controlled by a cytoplasmic repressor (RICHMOND 1965), synthesis of active repressor must be blocked and pre-existing repressor must be inactivated before penicillinase synthesis can occur, If the penicillinase repressor contains tryptophan, then tryptophan starvation should block the synthesis of the penicillinase repressor. Fur- thermore, if the penicillinase repressor has a relatively short half-life, then pre-existing penicillinase repressor would become inactivated (decay) during tryptophan starvation. Thus tryptophan starvation of S. aureus might, in theory, cause the induction of penicillinase synthesis and the accumulation of active penicillinase. As the data in Table 1 show (compare the specific activities in lines 1 and 21, tryptophan deprivation increases the basal level of penicillinase slightly (less than twofold). However, the addition of 5MT or 6MT to the culture medium of a tryptophan-starved auxotroph (Table 1) or 5MT to a tryptophan prototroph (Table 2), induces penicillinase synthesis much more effectively than tryptophan starvation. Either protein turnover provides tryptophan for the con-


1.40 0 . 4 5 0.50 0 .

T u r b i d i t y (535nm)

13

'5

FIGURE 10.-Penicillinase deinduction kinetics for CBAP-induced cells. Strain 655N (PI,,,) was obtained from a n enriched slant, and the cells were transferred to and grown on nutrient broth medium for 1 hr. The cells were collected, washed, and suspended in CAA medium and allowed to achieve exponential growth. CBAP (7.25 PM) and ferrous ion were added, and, after 15 (O), 30 (hexagon) or 45 ( A ) min in presence of the inducer, a large aliquot was removed, filtered, washed, and suspended in CAA medium. Samples (3 ml) were removed from each freshly-resuspended culture at zero-time and at 3-min intervals thereafter, and penicillinase activity was assayed.

tinued synthesis of the penicillinase repressor during tryptophan deprivation and this synthesis is in some way blocked by 5MT or 6MT, or 5MT and 6MT play an active role in the induction of penicillinase synthesis. Incorporation of 3H isoleucine in the presence or absence of 100 pg/mlSMT showed that 5MT reduces the rate of overall protein synthesis in a tryptophan prototroph by approximately 30% during a one-hour incubation (unpublished results) while tryptophan starvation reduces protein synthesis approximately 70% (Figure 1). Thus, net protein synthesis is reduced less by 5MT than by tryptophan starvation. On the basis of these observations, it can be argued that blocking protein synthesis is not the sole role played by 5MT in the induction of penicillinase synthesis. As shown in Figure 1, all tryptophan analog tested stimulate protein synthesis in tryptophan- starved cells. Since the 5MT used in these studies was free of tryptophan, one can conclude that this analog stimulates protein synthesis in the tryptophan-starved rells by becoming incorporated into cellular protein. Furthermore, data presented in Figure 4 indicate that the penicillinase repressor contains tryptophan, and it seems extremely likely, therefore, that 5MT becomes incorporated into the penicillinase repressor. Hence 5MT would appear i~ play at least two roles in the induction of penicillinase synthesis: (1 ) it reduces the rate of synthesis of tryptophan-containing proteins, including that of the penicillinase repressor; and (2) it becomes incorporated into protein and thus causes the newly synthesized peni-

14 J. IMSANDE

I I I

- 4 - E \ 0 10

.- : 3 - - - .- 0 .- 2 c

- P) Q

a

S 3

+-' 1 - .-

o 1 I I

T u r b i d i t y (535 nm) 0.40 0.45 0.50

FIGURE 11 .-Penicillinase deinduction kinetics for cells induced in the presence of CBAP and 5MT. Cells [655N(PI,,,)] were grown as described in Figure 10, and CBAP (7.25 p M ) and ferrous ion was added. After 15 min, an aliquot was removed, filtered, washed, and suspended in CAA medium. Crystalline 5MT (100 pg/ml final concentration) was added immediately to the remainder of the parent culture, and an aliquot was removed from it 15 and 30 min after the 5MT was added. These aliquots were filtered, washed, and suspended in CAA medium just as was the first aliquot. All three subcultures were sampled (3 ml) for penicillinase assay immediately after resuspension and at 3-min intervals thereafter. 0, 15-min induction by CBAP; hexagon, 15-min induction by CBAP followed by 15-min induction by both CBAP and 5MT; A, 15-min induction by CBAP followed by 30-min induction by both CBAP and 5MT.

cillinase repressor to be inactive. These two roles, in themselves, may be insuffi- cient, however, to account for the increased rate of penicillinase synthesis that occurs within the first hour following the addition of 5MT. This statement is based upon the fact that pre-existing penicillinase repressor would keep penicillinase synthesis repressed unless the penicillinase repressor has a short half-life. Therefore, either the repressor normally has a relatively short half-life (i.e., less than 10 min), or 5MT causes the effective half-life of pre-existing repressor to be shortened.

The induction of penicillinase as a result of the incorporation of 5MT and the slower rate of penicillinase deinduction during tryptophan starvation (Figure 4) provides strong biochemical evidence for the existence of a soluble penicillinase repressor. Also, these results indicate that penicillinase synthesis is regulated by negative control and not by positive control. Furthermore, these data (Figure 4) indicate that the penicillinase repressor contains tryptophan and, thus, explain why 5MT-induced cells deinduce rapidly when tryptophan is added to the culture medium.

A fully functional genetic repressor must contain two distinct sites. One site (A) on the repressor must be specific for the recognition of, and association with, the operator region on the DNA. The second site (e) must specifically recognize and interact with an effector or antirepressor. I t is generally not possible to assay


for a functional B site in the absence of an active A site; however, mutants that produce p-galactosidase repressor with an altered B site and an apparently normal A site have been described ( WILLSON et al. 1964). These mutants, the is class, fail to respond to the ,&galactosidase inducer. When 7-azatryptophan is added to the culture medium of S. aureus 5 minutes before the addition of the normal penicillinase inducer, CBAP, penicillinase synthesis is induced only slightly. Since 7-azatryptophan is incorporated into protein by S. aureus (Tables 1 and 3 and Figure 1 ) , one might speculate that 7-azatryptophan blocks penicillinase induction (see Figure 6) by creating a repressor that possesses an active A site but an inactive B site (i.e., an is-type repressor). If this speculation were correct, however. induced penicillinase synthesis should cease abruptly after the synthesis of a few molecules of the altered repressor. The deinduction kinetics presented in Figure 7 are inconsistent with this notion. Therefore, an iB-type repressor is not formed in the presence of 7-azatryptophan. How then does 7-azatryptophan block penicillinase induction (see Figure 6) ?

Genetic evidence shows that certain mutations, unlinked to the penicillinase plasmid, cause wild-type inducible type-I plasmids, but not type-I1 plasmids, to acquire a penicillinase constitutive phenotype ( COHEN and SWEENEY 1968). Similar genetic evidence shows that other mutations, unlinked to the penicillinase plasmid, cause wild-type inducible type-I1 plasmids, but not type-I plasmids, to display a penicillinase constitutive phenotype (COHEN, VERNON and SWEENEY 1970). It has been postulated thdt the second penicillinase regulatory gene directs the synthesis of a protein with the potential of inactivating the penicillinase repressor (IMSANDE 1970; IMSANDE, ZYSKIND and MILE 1972). Data have been presented that suggest this protein (i.e., the penicillinase antirepressor) must be synthesized de nouo in the presence of penicillin, or a penicillin derivative, to acquire the ability to inactivate the penicillinase repressor (IMSANDE, ZYSKIND and MILE 1972). Evidence for the inducibility of the penicillinase antirepressor could not be obtained; there€ore, it was deduced that the normal penicillinase inducer directs the conforma tion of the penicillinase antirepressor during its de novo synthesis. If 7-azatryptophan were to be incorporated into the penicillinase antirepressor, and deinduction studies with 5-methyltryptophan (Figures 10 and 11) or during tryptophan starvation (Figure 4) as well as induction studies during tryptophan starvation (Figure 5) indicate that the protein contains tryptophan, it may not be able to acquire an active conformation even in the presence of the authentic inducer. Thus, it is concluded that 7-azatryptophan blocks penicillinase induction by becoming incorporated into the proposed penicillinase antirepressor protein and thereby preventing the antirepressor from acquiring an active conformation. Thus the results presented in this communication confirm the general features of the proposed model for the regulation of penicillinase synthesis (IMSANDE, ZYSKIND and MILE 1972) and allow the model to be simplified as shown in Figure 12. According to this model, penicillinase induction proceeds by the following mechanism: growth of cells in the presence of CBAP, the most nearly gratuitous penicillinase inducer known

16 J. IMSANDE

Par Gene ( ch romosome)

7

I t -

(plasmid) Pen I Pen 0 Pen Z

(Parp. R I

FIGURE 12.-A working model for the regulation of penicillinase synthesis. Pen I: regulatory gene that specifies the cytoplasmic repressor (R). Pen 0: penicillinase operator. Pen Z: structural gene for penicillinase (Pen I, Pen 0, and Pen Z are normally carried on a plasmid). Par gene: regulatory gene, located on the host chromosome that specifies the penicillinase antirepressor, i.e., a protein that may inactivate the penicillinase repressor. NC: nascent chains of the penicillinase antirepressor. Reaction a: synthesis of the penicillinase antirepressor in absence of inducer generates the inactive conformation desigated Par,. Reaction b: synthesis in the presence of CBAP or penicillin produces the active conformation Par,. Reaction c: synthesis of the penicillinase antirepressor in the presence of 5-methyltryptophan or 5 MT plus CBAP produces the inactive conformation Par MT. Paro: inactive penicillinase antirepressor. Par,: active antirepressor, the conformation of which is directed by penicillin or CBAP. Par,,: inactive antirepressor, the conformation of which is altered by the incorporation of 5-methyltryptophan. RMT: inactive repressor that contains 5MT. Par, R: repressor-antirepressor complex. The formation of the complex inactivates the penicillinase repressor and hence penicillinase synthesis occurs.

to date, causes the newly synthesized antirepressor protein molecules, which seem to be formed constitutively, to acquire an active conformation (par,). This conformation prompts the antirepressor to interact with, and thereby inactivate, the penicillinase repressor (R) . Inactivation of the penicillinase repressor permits transcription of the penicillinase structural gene, and, subsequently, penicillinase synthesis occurs.

Although there is no definitive experimental evidence as yet for the involve- ment of an antirepressor in the regulation of cell division, this relationship can be readily conceptualized: ( 1 ) immediately following cell division (i.e., during the GI period) a daughter cell synthesizes a burst of a protein that represses the events responsible fo r thc initiation of cell division; (2) after chromosome repli- cation is completed (i.e., during the G, period) the cell synthesizes a burst of antirepressor that inactivates the repressor. Subsequently cell division occurs.


Thus an antirepressor could conceivably be employed in the control of cell division in both normal and neoplastic tissue.

The capable technical assistance of HYO-YOUNG CHOI is gratefully acknowledged.

LITERATURE CITED

AMBLER, R. P. and R. J. MEADWAY, 1969

BARLATI, S . and 0. CIFERRI, 1970

COHEN, S., E. G. VERNON and H. M. SWEENEY, 1970

Chemical structure of bacterial penicillinases. Nature

Incorporation of 5-methyl- and 5-hydroxytryptophan into the protein of Bacillus subtilis. .I. Bacterial. 101 : 166-172.

Differential derepression of staphylococcus plasmid and chromosomal penicillinase genes by a class of unlinked chromosomal mutations (R2-). J. Bacteriol. 103: 616-621.

COHEN, S. and H. M. SWEENEY, 1968 Constitutive penicillinase formation in Staphylococcus aureus owing to a mutation unlinked to the penicillinase plasmid. J. Bacteriol. 95: 1368- 1374.

DOOLITTLE, W. F. and CHARLES YANOFSKY, 1968 Mutants of Escherichia coli with an altered tryptophanyl-transfer ribonucleic acid synthetase. J. Bacteriol. 95 : 1283-1294.

hrs."E, JOHN, 1970 Regulation of penicillinase synthesis: evidence for a unified model. J. Bacteriol. 101 : 173-180.

IMsANDE, JOHN, J. w. ZYSKIND and IMRE MILE, 1972 Regulation of staphylococcal penicillinase synthesis. J. Bacteriol. 109: 122-133.

LARK, K. G., 1969 Incorporation of 5-methyltryptophan into the protein of Escherichia coli 15T-(555-7). J. Bacteriol. 97: 980-982.

LIU, T-Y and Y. H. CHANG, 1971 Hydrolysis of proteins with p-toluene-sulfonic acid. J. Biol. Chem. 246: 2842-2848.

MORSE, M. L., K. L. HILL, J. B. EGAN and W. HENGSTENBERG, 1968 Metabolism of lactose by Staphylococcus aureus and its genetic basis. J. Bacteriol. 95 : 2270-2274.

MOSTELLER, R. D. and CHARLES YANOFSKY, 1971 Evidence that tryptophanyl transfer ribonucleic acid is not the corepressor of the tryptophan operon of Escherichia coli. J. Bacteriol.

Incorporation d'analogues structuraux d'aminoacides dans les proteines bacteriennes au cours de leurs synthese in vivo. Biochim. Biophys. Acta 31 : 378-391.

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MUNIER, R. and G. N. COHEN, 1959

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PARDEE, A. B. and L. S. Prestidge, 1958

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RICHMOND, M. H., 1965

WILLSON, C., D. PERFUN, M. COHN, F. JACOB and J. MONOD, 1964.

ZYSKIND, J. W. and J. IMSANDE, 1972

Staphylococcus aureus. J. Gen. Microbiol. 33: 121-137.

bacteriophage. Biachim. Biophys. Acta 27: 330-344.

Iodometric assay of penicillinase. Nature 174: 1012-1013.

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Non-inducible mutants of the containing two distinct penicillinase plasmids. J. Bacteriol. 90: 370-374.

regulator gene in the "lactose" system of Escherichia coli. J. Mol. Biol. 8 : 582-592.

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Corresponding Editor: P. HARTMAN

is or · however, that 5mt is incorporated by tryptophan-starved e. coli (lark 1970; mosteller and...

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