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Factors influencing the synthesis of penicillinase by Micrococcus pyogenes
Factors Induencing the Synthesis of Penicillinase by Micrococcus pyogenesl Amedeo Bondi, Margaret de Saint Phalle, Joan Komblum and Albert G. Moat From
the Department
of Bacteriology,
Hahnemann Pennsylvania
Received
March
Medical
College,
Philadelphia,
15, 1954
INTRODUCTION The inactivation of penicillin by penicillinase with the resulting formation of penicilloic acid (1) has been studied by Foster (2) using Bacillus sublilis, by Henry and Housewright (3) and Pollock (4) using Bacillus cereus, and by Kirby (5) and Spink and Ferris (6) using Micrococcus pyogenes (Staphylococcus). In view of the general increase in the incidence of penicillin-resistant strains of M. pyogenes and their importance in clinical infections of man (7), it is surprising that further studies have not been carried out to elucidate factors influencing the production of penicillinase by this organism. This is particularily true since the resistance to penicillin of this organism is specifically related to its ability to produce this enzyme (6, 8). It was thought worth while, therefore, to study in further detail the formation of penicillinase by a resistant strain of M. pyogenes. For this study the authors have modified the manometric procedure used by Henry and Housewright (3) and by Pollock (4), which employs measurement of CO* evolved when the reaction is conducted in the presence of bicarbonate buffer. Some of the factors of importance in the synthesis of penicillinase by this organism are presented. MATERIALS AND METHODS Micrococcus pyogenes var. cillinase, was used throughout 1 This National
investigation Institutes
aureus, strain 7739, a culture the study. In all experiments
was supported, in part, by a research of Health, U. S. Public Health Service. 348
known to produce penithe source of the engrant,
EL!%, from
the
SYNTHESIS
OF PENICILLINASE
349
zyme was a resting cell suspension harvested generally from brain-heart infusion (BHI) broth cultures grown at 37°C. for 13-24 hr. in flasks aerated by shaking. After centrifugation the cells were washed once and resuspended in distilled water to give the desired concentration. The latter was determined turbidimetrically at 515 rnp on an Evelyn calorimeter and expressed as dry weight determined from a standard curve correlating milligrams dry weight with turbidity. The culture filtrate was discarded inasmuch as repeated tests showed that practically all the enzyme activity was found within the cells of M. pyogenes. By contrast, most of the activity of the enzyme produced by B. cereus is found to be extracellular (4). The intracellular nature of the enzyme did not affect measurement of the activity by the technique employed. Preliminary experiments showed that the penicillinase activity of a suspension of cells of M. pyogenes, strain 7739, could be measured in the Warburg apparatus at 37°C. at pH 7.0 in a COrbicarbonate buffer. The experimental methods, otherwise, were similar to those used by Henry and Housewright (3) and by Pollock (4). RESULTS
Effect of Substrate and Enzyme Concentration In order to ascertain whether the activity of the enzyme could be quantitated, the effect of varying substrate and enzyme concentrations was studied. The former was determined by measuring the reaction rate in the presenceof a seriesof concentrations of penicillin varying from 200 to 10,000 units. As shown in Fig. 1, the activity is typical of that of most enzyme reactions when the concentration of substrate is varied. In studying the effect of enzyme concentration on the reaction rate, and in all subsequent experiments, 10,000 units of the antibiotic was used routinely so that an excess of substrate would be present. The reaction rate obtained with various cell concentrations under these conditions is likewise shown in Fig. 1. A cell concentration of l-2 mg. was selected as the most suitable for subsequent studies. E$ect of Substrate on Enzyme Synthesis To obtain evidence as to the possible adaptive formation of penicillinase by M. pyogenessimilar to that described by Pollock (4) for B. cereus, two different experiments were run. In the first, the activity of cells harvested from BHI broth cultures to which penicillin had beenadded before inoculation to give concentrations of 1.0 and 10 units/ml. was compared with that of control cells grown in the absenceof the antibiotic. The cells in both cases were harvested after different periods of incubation. As shown in Table I, the penicillinase activity of cells grown in the presence of penicillin is essentialy the same as those grown in its absence.The
350
BONDI,
DE
SAINT
PHALLE,
120 -yl C02/HR/MG
90.
KORNBLUM
AND
MOAT
o/O--o
o/oR
.,./
3ovl 0
200
’
0
PENICILLIN ’ ’ ’
’ 5
x IO3 UNITS n a ’
.
*yl CO,/HR/MG
0
0
100 -
’ IO
/
/ OH0 0’
o-” 0
FIQ. 1. Effect of substrate
’
MG CELLS ’ ’ ’ 5
’
(DRY *
and cell concentration M. pyogenes.
WT) * ’
’ IO
on the penicillinase
activity
of
slight elevation in activity of cells grown in the presence of penicillin is believed to be the result of a stabilizing effect of substrate or its end products on the enzyme. This effect is quite apparent in the cells of the older cultures whose activity is lost more rapidly in the absenceof penicillin. In the second experiment, a method similar to that used by Pollock (4) in demonstrating the adaptive nature of the enzyme produced by B. cereuswas carried out. To flasks containing 5.0-ml. volumes of an unwashed ZO-hr. casein hydrolyzate-yeast extract broth culture of M. pyogenes,penicillin was added to give concentrations of 1, 100, and 1000 units/ml. The three flasks together with a control flask containing culture without penicillin were placed on a shaker at 37°C. Simultaneously, a culture of B. cereus N.R.R.L. B-569 known to adaptively produce
SYNTHESIS
OF
TABLE
of the
Effect
of Penicillin of Penicillinase
Addition
I
to Growth by M. Penicillinase
Penicillin
in growth U?dS/d.
medium
20
None 1 10
of Pretreatment cereus
of Cultures
Organism
upon the Production
activity, ~1. COz/hr./mg. Age of culture, kowrs 44
20.8 38.6 36.1
68
5.5 12.6 -
II
of Micrococcus
on the Adaptive Formation
pyogenes 7739 B. cereus N.R.R.L. B-569 M.
Medium
pyogenes
54.3 62.6 57.3
TABLE Effect
351
PENICILLINASE
NOIE
91 540
pyogenes and Bacillus of Penicillinase
Penicillinase activity, rJ. Cot/h?. Penicillin, w&s/ml 1 100
86 2620
79 3940
1000
118 3900
0 Added to 5.0-ml. volumes of casein hydrolyeate-yeast extract broth cultures of each organism and shaken for 1 hr. at 37°C. before measurement of activity.
penicillinase under the above conditions was treated in a similar fashion. At the end of 1 hr. the penicillinase activity of the contents of each flask was measured manometrically. As shown in Table II, the activity of B. cereus was greatly increased as a result of pretreatment with penicillin. Under the same condition, however, the activity of the cells of M. pyogenes was not significantly increased, indicating the inability of this organism to produce the enzyme adaptively. Even when longer intervals of pretreatment with penicillin were employed, essentially the same results were obtained. E$ect of Nutritional Conditions on Penicillinuse Synthesis
In studies designed to elucidate the relationship of various nutritional factors on enzyme formation, the activity of cells grown in various types of fluid media was measured. Included among the media studied were two defined media, amino acid (AA) No. 1 and AA No. 2, containing amino acids as sources of nitrogen, both of which are capable of supporting growth of the test strain. 2+3 Medium AA No. 1 represents the mini2 Amino acid No. 1 contains salts, glucose, thiamine, nicotinamide, biotin, glytine, cystine, leucine, arginine, proline, tryptophan, valine, glutamic acid, phenylalanine, and aspartic acid.
352
BONDI,
DE
SAINT
PHALLE,
TABLE
Penicillinase
KORNBLUM
AND
III
Production by Micrococcus pyogenes Media of Di$erent Composition Medium
No. No.
1). Vitamins 2). Vitamins
in
Fluid
Penicillinase rrl. COzlhr./mng.
Brain heart infusion Trypticase soy Meat extract Casein hydrolyzate Amino acid No. la Amino acid No. 2” Q (A.A. b (A.A.
MOAT
53 39 49 115 140 56
and 10 amino and 19 amino
acids
(see text).
acids (seetext).
ma1 requirements for growth of this strain under aerated conditions. The cells harvested from each medium after 20 hr. growth were standardized turbidimetrically to compensate for growth differences. The activity of 1 mg. of cells from each medium was tested and compared. As shown in Table III, the activity of cells grown in the various media varied considerably. Cells harvested from the casein hydrolyzate medium were considerably more active than those grown in more complex media. Of particular note is the significantly lower activity of cells grown in medium AA No. 2 as compared with the higher activity in cells from medium AA No. 1, despite the fact that the amount of growth in AA No. 2 is slightly greater. These observations suggest the possibility that certain amino acids may inhibit synthesis of penicillinase. DISCUSSION
Although Kirby (5) and Spink and Ferris (6) studied some of the characteristics of penicillinase produced by M. pyogenes,they did not approach the problem of factors influencing its formation. We were able to show that adaptive formation of staphylococcal penicillinase does not occur under conditions which permit adaptive formation of the enzyme by B. cereus (4). The obvious conclusion is that the enzyme produced by this organism is a constitutive enzyme even under well-defined nutritional conditions. This might indicate that it has an essential role in the organism’s metabolism although no experimental proof is available to substantiate such a claim. The only other major difference between a Amino hydroxyproline, methionine.
acid No. 2 contains in addition to the ingredients in AA No. 1, alanine, histidine, iysine, isoleucine, serine, threonine, tyrosine, and
SYNTHESIS
OF
PENICILLINASE
353
the penicillinase produced by the two organisms is the intracellular location of that of the staphylococcus in contrast to the extracellular location of that of B. cereus. It does not appear likely, however, that this difference would account for the inability of the M. pyogenes to produce the enzyme adaptively. Of considerable interest to US is the observation that certain amino acids may suppress enzyme production by this organism. In view of the major role of penicillinase in the resistance of staphylococci to penicillin (6, S), such interference in enzyme production may prove to be a potential weapon in rendering this organism more susceptible to the antibiotic. Further studies along these lines are indicated. SUMMARY
Some of the factors influencing synthesis of penicillinase by M. pyoare described. Adaptive formation of the enzyme by this organism was not observed under conditions permitting adaptive formation by BaciZZuscereus. Penicillinase activity of cells harvested from various media varies considerably suggesting the possibility that certain agents may suppress enzyme formation. genes
REFERENCES of Penicillin,” 1. ABRAHAM, E. P., BAKER, W., BOON, W. R., et al., “The Chemistry Chap. 2. Princeton University Press, Princeton, N. J., 1949. 2. FOSTER, J. W., Science 101, 205 (1945). 3. HENRY, R. J., AND HOUSEWRIGHT, R. D., J. Bio.?. Chem. 167, 559 (1947). 4. POLLOCK, M. R., Brit. J. Exptl. Pathol. 31, 739 (1950). 5. KIRBY, W. M. M., J. Clin. Invest. 24, 170 (1945). 6. SPINK. W. W., AND FERRIS, V., J. Clin. Invest. 26, 379 (1947). 7. BONDI, A., PFAFF, F., FREE, E., AND SWERLICK, R., Am. J. Public Health 44, 789 (1954). 8. BONDI, A., AND DIETZ, C., Proc. Sot. Ezptl. Biol. Med. 80, 55 (1945).
the Department
of Bacteriology,
Hahnemann Pennsylvania
Received
March
Medical
College,
Philadelphia,
15, 1954
INTRODUCTION The inactivation of penicillin by penicillinase with the resulting formation of penicilloic acid (1) has been studied by Foster (2) using Bacillus sublilis, by Henry and Housewright (3) and Pollock (4) using Bacillus cereus, and by Kirby (5) and Spink and Ferris (6) using Micrococcus pyogenes (Staphylococcus). In view of the general increase in the incidence of penicillin-resistant strains of M. pyogenes and their importance in clinical infections of man (7), it is surprising that further studies have not been carried out to elucidate factors influencing the production of penicillinase by this organism. This is particularily true since the resistance to penicillin of this organism is specifically related to its ability to produce this enzyme (6, 8). It was thought worth while, therefore, to study in further detail the formation of penicillinase by a resistant strain of M. pyogenes. For this study the authors have modified the manometric procedure used by Henry and Housewright (3) and by Pollock (4), which employs measurement of CO* evolved when the reaction is conducted in the presence of bicarbonate buffer. Some of the factors of importance in the synthesis of penicillinase by this organism are presented. MATERIALS AND METHODS Micrococcus pyogenes var. cillinase, was used throughout 1 This National
investigation Institutes
aureus, strain 7739, a culture the study. In all experiments
was supported, in part, by a research of Health, U. S. Public Health Service. 348
known to produce penithe source of the engrant,
EL!%, from
the
SYNTHESIS
OF PENICILLINASE
349
zyme was a resting cell suspension harvested generally from brain-heart infusion (BHI) broth cultures grown at 37°C. for 13-24 hr. in flasks aerated by shaking. After centrifugation the cells were washed once and resuspended in distilled water to give the desired concentration. The latter was determined turbidimetrically at 515 rnp on an Evelyn calorimeter and expressed as dry weight determined from a standard curve correlating milligrams dry weight with turbidity. The culture filtrate was discarded inasmuch as repeated tests showed that practically all the enzyme activity was found within the cells of M. pyogenes. By contrast, most of the activity of the enzyme produced by B. cereus is found to be extracellular (4). The intracellular nature of the enzyme did not affect measurement of the activity by the technique employed. Preliminary experiments showed that the penicillinase activity of a suspension of cells of M. pyogenes, strain 7739, could be measured in the Warburg apparatus at 37°C. at pH 7.0 in a COrbicarbonate buffer. The experimental methods, otherwise, were similar to those used by Henry and Housewright (3) and by Pollock (4). RESULTS
Effect of Substrate and Enzyme Concentration In order to ascertain whether the activity of the enzyme could be quantitated, the effect of varying substrate and enzyme concentrations was studied. The former was determined by measuring the reaction rate in the presenceof a seriesof concentrations of penicillin varying from 200 to 10,000 units. As shown in Fig. 1, the activity is typical of that of most enzyme reactions when the concentration of substrate is varied. In studying the effect of enzyme concentration on the reaction rate, and in all subsequent experiments, 10,000 units of the antibiotic was used routinely so that an excess of substrate would be present. The reaction rate obtained with various cell concentrations under these conditions is likewise shown in Fig. 1. A cell concentration of l-2 mg. was selected as the most suitable for subsequent studies. E$ect of Substrate on Enzyme Synthesis To obtain evidence as to the possible adaptive formation of penicillinase by M. pyogenessimilar to that described by Pollock (4) for B. cereus, two different experiments were run. In the first, the activity of cells harvested from BHI broth cultures to which penicillin had beenadded before inoculation to give concentrations of 1.0 and 10 units/ml. was compared with that of control cells grown in the absenceof the antibiotic. The cells in both cases were harvested after different periods of incubation. As shown in Table I, the penicillinase activity of cells grown in the presence of penicillin is essentialy the same as those grown in its absence.The
350
BONDI,
DE
SAINT
PHALLE,
120 -yl C02/HR/MG
90.
KORNBLUM
AND
MOAT
o/O--o
o/oR
.,./
3ovl 0
200
’
0
PENICILLIN ’ ’ ’
’ 5
x IO3 UNITS n a ’
.
*yl CO,/HR/MG
0
0
100 -
’ IO
/
/ OH0 0’
o-” 0
FIQ. 1. Effect of substrate
’
MG CELLS ’ ’ ’ 5
’
(DRY *
and cell concentration M. pyogenes.
WT) * ’
’ IO
on the penicillinase
activity
of
slight elevation in activity of cells grown in the presence of penicillin is believed to be the result of a stabilizing effect of substrate or its end products on the enzyme. This effect is quite apparent in the cells of the older cultures whose activity is lost more rapidly in the absenceof penicillin. In the second experiment, a method similar to that used by Pollock (4) in demonstrating the adaptive nature of the enzyme produced by B. cereuswas carried out. To flasks containing 5.0-ml. volumes of an unwashed ZO-hr. casein hydrolyzate-yeast extract broth culture of M. pyogenes,penicillin was added to give concentrations of 1, 100, and 1000 units/ml. The three flasks together with a control flask containing culture without penicillin were placed on a shaker at 37°C. Simultaneously, a culture of B. cereus N.R.R.L. B-569 known to adaptively produce
SYNTHESIS
OF
TABLE
of the
Effect
of Penicillin of Penicillinase
Addition
I
to Growth by M. Penicillinase
Penicillin
in growth U?dS/d.
medium
20
None 1 10
of Pretreatment cereus
of Cultures
Organism
upon the Production
activity, ~1. COz/hr./mg. Age of culture, kowrs 44
20.8 38.6 36.1
68
5.5 12.6 -
II
of Micrococcus
on the Adaptive Formation
pyogenes 7739 B. cereus N.R.R.L. B-569 M.
Medium
pyogenes
54.3 62.6 57.3
TABLE Effect
351
PENICILLINASE
NOIE
91 540
pyogenes and Bacillus of Penicillinase
Penicillinase activity, rJ. Cot/h?. Penicillin, w&s/ml 1 100
86 2620
79 3940
1000
118 3900
0 Added to 5.0-ml. volumes of casein hydrolyeate-yeast extract broth cultures of each organism and shaken for 1 hr. at 37°C. before measurement of activity.
penicillinase under the above conditions was treated in a similar fashion. At the end of 1 hr. the penicillinase activity of the contents of each flask was measured manometrically. As shown in Table II, the activity of B. cereus was greatly increased as a result of pretreatment with penicillin. Under the same condition, however, the activity of the cells of M. pyogenes was not significantly increased, indicating the inability of this organism to produce the enzyme adaptively. Even when longer intervals of pretreatment with penicillin were employed, essentially the same results were obtained. E$ect of Nutritional Conditions on Penicillinuse Synthesis
In studies designed to elucidate the relationship of various nutritional factors on enzyme formation, the activity of cells grown in various types of fluid media was measured. Included among the media studied were two defined media, amino acid (AA) No. 1 and AA No. 2, containing amino acids as sources of nitrogen, both of which are capable of supporting growth of the test strain. 2+3 Medium AA No. 1 represents the mini2 Amino acid No. 1 contains salts, glucose, thiamine, nicotinamide, biotin, glytine, cystine, leucine, arginine, proline, tryptophan, valine, glutamic acid, phenylalanine, and aspartic acid.
352
BONDI,
DE
SAINT
PHALLE,
TABLE
Penicillinase
KORNBLUM
AND
III
Production by Micrococcus pyogenes Media of Di$erent Composition Medium
No. No.
1). Vitamins 2). Vitamins
in
Fluid
Penicillinase rrl. COzlhr./mng.
Brain heart infusion Trypticase soy Meat extract Casein hydrolyzate Amino acid No. la Amino acid No. 2” Q (A.A. b (A.A.
MOAT
53 39 49 115 140 56
and 10 amino and 19 amino
acids
(see text).
acids (seetext).
ma1 requirements for growth of this strain under aerated conditions. The cells harvested from each medium after 20 hr. growth were standardized turbidimetrically to compensate for growth differences. The activity of 1 mg. of cells from each medium was tested and compared. As shown in Table III, the activity of cells grown in the various media varied considerably. Cells harvested from the casein hydrolyzate medium were considerably more active than those grown in more complex media. Of particular note is the significantly lower activity of cells grown in medium AA No. 2 as compared with the higher activity in cells from medium AA No. 1, despite the fact that the amount of growth in AA No. 2 is slightly greater. These observations suggest the possibility that certain amino acids may inhibit synthesis of penicillinase. DISCUSSION
Although Kirby (5) and Spink and Ferris (6) studied some of the characteristics of penicillinase produced by M. pyogenes,they did not approach the problem of factors influencing its formation. We were able to show that adaptive formation of staphylococcal penicillinase does not occur under conditions which permit adaptive formation of the enzyme by B. cereus (4). The obvious conclusion is that the enzyme produced by this organism is a constitutive enzyme even under well-defined nutritional conditions. This might indicate that it has an essential role in the organism’s metabolism although no experimental proof is available to substantiate such a claim. The only other major difference between a Amino hydroxyproline, methionine.
acid No. 2 contains in addition to the ingredients in AA No. 1, alanine, histidine, iysine, isoleucine, serine, threonine, tyrosine, and
SYNTHESIS
OF
PENICILLINASE
353
the penicillinase produced by the two organisms is the intracellular location of that of the staphylococcus in contrast to the extracellular location of that of B. cereus. It does not appear likely, however, that this difference would account for the inability of the M. pyogenes to produce the enzyme adaptively. Of considerable interest to US is the observation that certain amino acids may suppress enzyme production by this organism. In view of the major role of penicillinase in the resistance of staphylococci to penicillin (6, S), such interference in enzyme production may prove to be a potential weapon in rendering this organism more susceptible to the antibiotic. Further studies along these lines are indicated. SUMMARY
Some of the factors influencing synthesis of penicillinase by M. pyoare described. Adaptive formation of the enzyme by this organism was not observed under conditions permitting adaptive formation by BaciZZuscereus. Penicillinase activity of cells harvested from various media varies considerably suggesting the possibility that certain agents may suppress enzyme formation. genes
REFERENCES of Penicillin,” 1. ABRAHAM, E. P., BAKER, W., BOON, W. R., et al., “The Chemistry Chap. 2. Princeton University Press, Princeton, N. J., 1949. 2. FOSTER, J. W., Science 101, 205 (1945). 3. HENRY, R. J., AND HOUSEWRIGHT, R. D., J. Bio.?. Chem. 167, 559 (1947). 4. POLLOCK, M. R., Brit. J. Exptl. Pathol. 31, 739 (1950). 5. KIRBY, W. M. M., J. Clin. Invest. 24, 170 (1945). 6. SPINK. W. W., AND FERRIS, V., J. Clin. Invest. 26, 379 (1947). 7. BONDI, A., PFAFF, F., FREE, E., AND SWERLICK, R., Am. J. Public Health 44, 789 (1954). 8. BONDI, A., AND DIETZ, C., Proc. Sot. Ezptl. Biol. Med. 80, 55 (1945).