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On growth and nutritional comparisons of a group A streptococcus before and after cell wall removal
ARCHIVES
On
OF
BIOCHEMISTRY
Growth
and
AND
BIOPHYSICS
Nutritional before
From
the Research
(1964)
Comparisons and
CHARLES
3266328
106,
after
PANOS
Laboratories, Center,
Cell
Received
Wall
A Streptococcus
Removal’
LILLIAN
M. HYNES
of Biochemistry,
The Albert
AND
Department Philadelphia,
of a Group
Einstein
Medical
Pennsylvania
November
4, 1963
Major differences in the vitamin requirements were not detected upon conversion of a coccus to a stable L form, i.e., permanent loss of cell wall. A purified protein has been found that successfully replaces the heterogeneous and more complex horse serum hitherto required as a necessary ingredient for growth of the coccal L form. A medium of low vitamin and low lipid content has been compounded that may prove useful for further nutritional as well as lipid studies of reproducing coccal L forms lacking a cell wall. INTRODUCTION
Group A streptococci may lose their ability to continue cell wall biosynthesis after treatment with penicillin and the employment of proper osmotic conditions. The resulting osmotically fragile, viable, filterable, and reproducing forms have been termed “L forms.” It has also been demonstrated that when a stable L form is derived from a group A streptococcus, the permanent loss of cell wall biosynthetic capabilities is accompanied by concomitant alterations in carbohydrate metabolism (1) and chemical composition (2), and results in the accumulation of a UDP-muramic acid-peptide (3) which has been implicated as a possible cell wall precursor. These findings have strengthened the belief that an L form may not be merely a bacterial cell lacking its characteristic rigid cell wall. A major difficulty in studying L forms derived from group A streptococci has been the necessity to grow them incomplex, ill-defined media which contain, in addition to extracts of natural products, large quantities of horse serum. Such media have prevented detailed nutritional studies and have given rise to cellular lipid analyses which mimic the lipid the
1 Supported USPHS.
by
funds
from
grant
E-4495
from
content of the complex growth medium. Therefore, attempts were initiated to grow and compare the vitamin requirements of a group A streptococcus and an L form derived from it in a medium of low vitamin and lipid content. MATERIALS
METHODS
The streptococcus and L form are the same as those used in earlier studies (1, 3). Broth cultures of these organisms were maintained in complex media containing 10% horse serum (v/v) and in the experimental medium fortified with 1.4 g/100 ml. brucella broth (Albimi Labs.). Cultures were incubated overnight at 36°C. and stored at 5°C. until needed. The COM-SYN-6 medium of Ginsburg and Grossowicz (4) was used with the following modifications: the sodium phosphate concentration was reduced by one-half, and sodium acetate and Lcysteine (100 mg. each2), adenine sulfate (1 mg.), and enzymatic “vitamin-free” casein hydrolysate (10 ml. Nutritional Biochemicals) were added. Vitamin requirements were determined essentially as described elsewhere (5). In addition to the COM-SYNB vitamin content, the effect of omission of pyridoxine and nicotinamide (1 mg. each), p-aminobenzoic acid (0.08 me.), B12 (5 X 1O-6 mg.) and folic acid (3 X 10m5 mg.) upon growth was also examined. 2.3 g. of a brucella 2 All concentrations experimental medium.
326
AND
expressed
per
100 ml.
of
\‘ITAMIN
REQUIREMENTS
OF
broth “vitamin-free” fraction which was prepared by repeated passage of an aqueous solution over activated charcoal (until O.D. = 0.190 at 400 x with a Coleman model 14 spectrophotometer resulted; distilled water as blank) was required for growth of either organism. For the osmotically fragile L form, sodium chloride was increased to 3 g. and bovine plasma albumin (0.8 g.), fraction V (Armour), replaced the horse serum requirement for the L form. Test media were dispensed in loml. amounts in X-ml. Erlenmeyer flasks, incubated for 16 hours at 36”C., and growth determined with the Coleman spectrophot,ometer at 650 X. Sterilization by heat (less albumin) or filtration yielded similar results. Uninoculated media served as controls. Inocula were prepared from logarithmic cultures from 10 ml. of fortihe 1 experimental medium by centrifugation and washing five times with 10 ml. of physiological saline (coccus) or 5 ml. of 3y0 sodium chloride solution (L form) at 2000 rpm. Aliquotjs of the resulting almost clear cocc:rl (0.3 ml.) or L form (0.5 ml.) suspensions served as inocula for each experimerltal flask. For total lipid content, 100 ml. of each growth medium, in duplicate, was lyophilized and saponified; the nonsaponifiable matter was removed, and the free fatty acids were recovered as mentioned elsewhere (6).
GROUP
37
A STREPTOCOCCUS RESULTS
AXD
DISCUSSION
Under these experimental conditions, both organisms had an absolute requirement for riboflavin, and equal growth responses were obtained in the absence of t’he other vitamins
Grams per lOOmI of Experimental FIG. 1. Typical growth amounts of “vitamin-free” broth for streptococcus
response to increasing fraction from brucella and derived L form.
TBBLE GROWTH
ABILITY
FORM
IN
II
OF THE
COCCIJS
EXPERIMENTAL
VITAMIN REQUIREMENTS STREPTOCOCCUS AXD EXPERIMENTAL
GROWTH
IN
PARENT THE
MEDIUM Growth”
Experimental
medium
Complete Less all added vitamins Less biotin Less nicotinamide Less nicotinic acid Less pantothenate Less pyridoxal Less pyridoxine Less thiamine Less p-aminobenzoic acid Less Bit Less folic acid Less riboflavin Only riboflavin Complex medium with horse serum a Expressed as the mean readings of four determinations
L-form
COCCUS
OF THE
of
streptomxCUS
L-form
25.0 94.0 27.0 24.1 25.0 21.1 24.1 24.1 25.9 26.0 24.0 25.0 97.0 19.5 11.8
37.0 83.0 40.0 36.2 54.0 47.0 48.0 38.9 42.9 36.0 38.2 37.8 82.0 36.8 27.9
the calorimeter (%7,T).
Complex with horse serum Experiment&
1.13
0.75
12.0
27.9
0.65
0.42
25.0
37.0
TABLE FATTY
ACID
VARIOUS
Medium
with
organism
L
form
and
III CONTENT
GROWTH
OF THE
MEIXA
used
Complex with horse serum for streptococcus and L form Experimental with albumin for L form Experimental without albumin for streptococcus a Average * Expressed
L-form
COCCUS
~1By turbidity and viable count. * By turbidity (702’). c With and without albumin for coccal growth, respectively.
TOTAL
L
Maximum growth response*
I L FORM
AND
MEDIA
Medium
TABLE
Medium
of six titrations. as stearic acid.
5.50
15.6
0.96
2.7
0.89
2.5
328
PANOS
AND
tested (Table I). Growth of the coccus and L form in the presence of riboflavin and the absence of nicotinic acid, pantothenate, or pyridoxal was less than that with riboflavin alone. A requirement for nicotinic acid and pantothenate, as well as for riboflavin, as reported by Ginsburg and Grossowicz (4) for three strains of group A streptococci in a chemically defined growth medium, was not observed. Attempts to grow our organisms in such a chemically defined medium were unsuccessful. Figure 1 demonstrates the necessity for the addition of a crude “vitamin-free” fraction from brucella broth for growth. Likewise, Kantor and Cole (7) added peptone dialysate to a nonprotein semisynthetic medium to achieve growth of a group C streptococcus. Slade et al. (8) reported the growth-stimulating effects of a crude peptide material (CR14A) from pancreatic casein for a group A, type 3 streptococcus. This peptide material was isolated but found to be without stimulating or growth-promoting activity for this type 12 coccus and derived L form. Bovine plasma albumin (fraction V) successfully replaces horse serum for growth of the L form in experimental and complex media. Neither albumin nor horse serum is required for growth of the parent coccus. Smith and Boughton (9) found that fraction V bovine albumin, at a level of 5 mg. per milliliter, promoted growth of a PPLO (strain 07), but that this enhancement disappeared upon further purification of the albumin; indicating the presenceof a growth promoting contaminant. In contrast to these findings, no decreasein the growth response of the L form was observed when highly purified crystallized bovine plasma albumin (CBA, Armour Tech. Dept.) replaced fraction V albumin both at levels of 8 mg per milliliter. The lipid content of CBA was 0.1 ‘Z as stearic acid (Armour, personal communication). Below this concentration, both CBA and fraction V preparations failed to support maximal growth. It is apparent that the growth rates of both cultures, in their respective experi-
HYNES
mental media, are approximately one-half of those obtained in the complex medium containing horse serum (Table II). However, although the growth rates are reduced, the maximal growth levels obtained are nearly comparable. The growth rate was not decreasedin the complex medium when CBA or fraction V albumin replaced horse serum for either organism. Since growth of the parent coccus is not dependent upon the presenceOY absence of albumin or horse serum (Table II), it is probable that the decreasedgrowth rates in the experimental media are due to other nutritive essentials removed or reduced during preparation of the crudefraction ingredient (Fig. 1). The complete experimental media for the L form and streptococcus supported growth for twenty consecutive transfers when discontinued. Table III tabulates the results of the total lipid content of the complex and experimental media used for growth of these organisms. It was possible to appreciably decrease the fatty acid content by replacing horse serum with fraction V albumin. Replacing this albumin by CBA yielded identical results. Apparently, albumin is not significantly contributing to the low lipid content of the experimental growth media. Therefore, the fatty acid content found is probably due to the two undefined medium ingredients : crude brucella broth fraction and/or enzymatic casein hydrolysate. REFERENCES 1. PANOS, 2. PANOS,
J. A.,
C., J. Racteriol. C., BARKULIS,
J. Bacterial.
3. EDWARDS, J., 1202 (1962).
AND
84, 921 (1962). S.
S.,
AND
HAYASHI,
78, 863 (1959). PANOS, C., J. Bacterial.
84,
I., AND GROSSOWICZ, N., Proc. Sot. Biol. Med. 96, 108 (1957). 5. O’LEARY, W. M., PANOS, C., AND HELZ, G. E., J. Bacterial. 72, 673 (1956). 6. O’LEARY, W. M., J. BacterioE. 84, 967 (1962). 7. KANT-OR, F. S., AND COLE, R. M., J. Exptl. Med. 112, 77 (1960). 8. SLADE, H. D., KNOX, G. A., AND SLAMP, W. C., J. Bacterial. 62, 669 (1951). 9. SMITH, P. F., AND BOUGHTON, J. E., J. Bacteriol. 80, 851 (1960). 4.
GINSBURG,
Exptl.
On
OF
BIOCHEMISTRY
Growth
and
AND
BIOPHYSICS
Nutritional before
From
the Research
(1964)
Comparisons and
CHARLES
3266328
106,
after
PANOS
Laboratories, Center,
Cell
Received
Wall
A Streptococcus
Removal’
LILLIAN
M. HYNES
of Biochemistry,
The Albert
AND
Department Philadelphia,
of a Group
Einstein
Medical
Pennsylvania
November
4, 1963
Major differences in the vitamin requirements were not detected upon conversion of a coccus to a stable L form, i.e., permanent loss of cell wall. A purified protein has been found that successfully replaces the heterogeneous and more complex horse serum hitherto required as a necessary ingredient for growth of the coccal L form. A medium of low vitamin and low lipid content has been compounded that may prove useful for further nutritional as well as lipid studies of reproducing coccal L forms lacking a cell wall. INTRODUCTION
Group A streptococci may lose their ability to continue cell wall biosynthesis after treatment with penicillin and the employment of proper osmotic conditions. The resulting osmotically fragile, viable, filterable, and reproducing forms have been termed “L forms.” It has also been demonstrated that when a stable L form is derived from a group A streptococcus, the permanent loss of cell wall biosynthetic capabilities is accompanied by concomitant alterations in carbohydrate metabolism (1) and chemical composition (2), and results in the accumulation of a UDP-muramic acid-peptide (3) which has been implicated as a possible cell wall precursor. These findings have strengthened the belief that an L form may not be merely a bacterial cell lacking its characteristic rigid cell wall. A major difficulty in studying L forms derived from group A streptococci has been the necessity to grow them incomplex, ill-defined media which contain, in addition to extracts of natural products, large quantities of horse serum. Such media have prevented detailed nutritional studies and have given rise to cellular lipid analyses which mimic the lipid the
1 Supported USPHS.
by
funds
from
grant
E-4495
from
content of the complex growth medium. Therefore, attempts were initiated to grow and compare the vitamin requirements of a group A streptococcus and an L form derived from it in a medium of low vitamin and lipid content. MATERIALS
METHODS
The streptococcus and L form are the same as those used in earlier studies (1, 3). Broth cultures of these organisms were maintained in complex media containing 10% horse serum (v/v) and in the experimental medium fortified with 1.4 g/100 ml. brucella broth (Albimi Labs.). Cultures were incubated overnight at 36°C. and stored at 5°C. until needed. The COM-SYN-6 medium of Ginsburg and Grossowicz (4) was used with the following modifications: the sodium phosphate concentration was reduced by one-half, and sodium acetate and Lcysteine (100 mg. each2), adenine sulfate (1 mg.), and enzymatic “vitamin-free” casein hydrolysate (10 ml. Nutritional Biochemicals) were added. Vitamin requirements were determined essentially as described elsewhere (5). In addition to the COM-SYNB vitamin content, the effect of omission of pyridoxine and nicotinamide (1 mg. each), p-aminobenzoic acid (0.08 me.), B12 (5 X 1O-6 mg.) and folic acid (3 X 10m5 mg.) upon growth was also examined. 2.3 g. of a brucella 2 All concentrations experimental medium.
326
AND
expressed
per
100 ml.
of
\‘ITAMIN
REQUIREMENTS
OF
broth “vitamin-free” fraction which was prepared by repeated passage of an aqueous solution over activated charcoal (until O.D. = 0.190 at 400 x with a Coleman model 14 spectrophotometer resulted; distilled water as blank) was required for growth of either organism. For the osmotically fragile L form, sodium chloride was increased to 3 g. and bovine plasma albumin (0.8 g.), fraction V (Armour), replaced the horse serum requirement for the L form. Test media were dispensed in loml. amounts in X-ml. Erlenmeyer flasks, incubated for 16 hours at 36”C., and growth determined with the Coleman spectrophot,ometer at 650 X. Sterilization by heat (less albumin) or filtration yielded similar results. Uninoculated media served as controls. Inocula were prepared from logarithmic cultures from 10 ml. of fortihe 1 experimental medium by centrifugation and washing five times with 10 ml. of physiological saline (coccus) or 5 ml. of 3y0 sodium chloride solution (L form) at 2000 rpm. Aliquotjs of the resulting almost clear cocc:rl (0.3 ml.) or L form (0.5 ml.) suspensions served as inocula for each experimerltal flask. For total lipid content, 100 ml. of each growth medium, in duplicate, was lyophilized and saponified; the nonsaponifiable matter was removed, and the free fatty acids were recovered as mentioned elsewhere (6).
GROUP
37
A STREPTOCOCCUS RESULTS
AXD
DISCUSSION
Under these experimental conditions, both organisms had an absolute requirement for riboflavin, and equal growth responses were obtained in the absence of t’he other vitamins
Grams per lOOmI of Experimental FIG. 1. Typical growth amounts of “vitamin-free” broth for streptococcus
response to increasing fraction from brucella and derived L form.
TBBLE GROWTH
ABILITY
FORM
IN
II
OF THE
COCCIJS
EXPERIMENTAL
VITAMIN REQUIREMENTS STREPTOCOCCUS AXD EXPERIMENTAL
GROWTH
IN
PARENT THE
MEDIUM Growth”
Experimental
medium
Complete Less all added vitamins Less biotin Less nicotinamide Less nicotinic acid Less pantothenate Less pyridoxal Less pyridoxine Less thiamine Less p-aminobenzoic acid Less Bit Less folic acid Less riboflavin Only riboflavin Complex medium with horse serum a Expressed as the mean readings of four determinations
L-form
COCCUS
OF THE
of
streptomxCUS
L-form
25.0 94.0 27.0 24.1 25.0 21.1 24.1 24.1 25.9 26.0 24.0 25.0 97.0 19.5 11.8
37.0 83.0 40.0 36.2 54.0 47.0 48.0 38.9 42.9 36.0 38.2 37.8 82.0 36.8 27.9
the calorimeter (%7,T).
Complex with horse serum Experiment&
1.13
0.75
12.0
27.9
0.65
0.42
25.0
37.0
TABLE FATTY
ACID
VARIOUS
Medium
with
organism
L
form
and
III CONTENT
GROWTH
OF THE
MEIXA
used
Complex with horse serum for streptococcus and L form Experimental with albumin for L form Experimental without albumin for streptococcus a Average * Expressed
L-form
COCCUS
~1By turbidity and viable count. * By turbidity (702’). c With and without albumin for coccal growth, respectively.
TOTAL
L
Maximum growth response*
I L FORM
AND
MEDIA
Medium
TABLE
Medium
of six titrations. as stearic acid.
5.50
15.6
0.96
2.7
0.89
2.5
328
PANOS
AND
tested (Table I). Growth of the coccus and L form in the presence of riboflavin and the absence of nicotinic acid, pantothenate, or pyridoxal was less than that with riboflavin alone. A requirement for nicotinic acid and pantothenate, as well as for riboflavin, as reported by Ginsburg and Grossowicz (4) for three strains of group A streptococci in a chemically defined growth medium, was not observed. Attempts to grow our organisms in such a chemically defined medium were unsuccessful. Figure 1 demonstrates the necessity for the addition of a crude “vitamin-free” fraction from brucella broth for growth. Likewise, Kantor and Cole (7) added peptone dialysate to a nonprotein semisynthetic medium to achieve growth of a group C streptococcus. Slade et al. (8) reported the growth-stimulating effects of a crude peptide material (CR14A) from pancreatic casein for a group A, type 3 streptococcus. This peptide material was isolated but found to be without stimulating or growth-promoting activity for this type 12 coccus and derived L form. Bovine plasma albumin (fraction V) successfully replaces horse serum for growth of the L form in experimental and complex media. Neither albumin nor horse serum is required for growth of the parent coccus. Smith and Boughton (9) found that fraction V bovine albumin, at a level of 5 mg. per milliliter, promoted growth of a PPLO (strain 07), but that this enhancement disappeared upon further purification of the albumin; indicating the presenceof a growth promoting contaminant. In contrast to these findings, no decreasein the growth response of the L form was observed when highly purified crystallized bovine plasma albumin (CBA, Armour Tech. Dept.) replaced fraction V albumin both at levels of 8 mg per milliliter. The lipid content of CBA was 0.1 ‘Z as stearic acid (Armour, personal communication). Below this concentration, both CBA and fraction V preparations failed to support maximal growth. It is apparent that the growth rates of both cultures, in their respective experi-
HYNES
mental media, are approximately one-half of those obtained in the complex medium containing horse serum (Table II). However, although the growth rates are reduced, the maximal growth levels obtained are nearly comparable. The growth rate was not decreasedin the complex medium when CBA or fraction V albumin replaced horse serum for either organism. Since growth of the parent coccus is not dependent upon the presenceOY absence of albumin or horse serum (Table II), it is probable that the decreasedgrowth rates in the experimental media are due to other nutritive essentials removed or reduced during preparation of the crudefraction ingredient (Fig. 1). The complete experimental media for the L form and streptococcus supported growth for twenty consecutive transfers when discontinued. Table III tabulates the results of the total lipid content of the complex and experimental media used for growth of these organisms. It was possible to appreciably decrease the fatty acid content by replacing horse serum with fraction V albumin. Replacing this albumin by CBA yielded identical results. Apparently, albumin is not significantly contributing to the low lipid content of the experimental growth media. Therefore, the fatty acid content found is probably due to the two undefined medium ingredients : crude brucella broth fraction and/or enzymatic casein hydrolysate. REFERENCES 1. PANOS, 2. PANOS,
J. A.,
C., J. Racteriol. C., BARKULIS,
J. Bacterial.
3. EDWARDS, J., 1202 (1962).
AND
84, 921 (1962). S.
S.,
AND
HAYASHI,
78, 863 (1959). PANOS, C., J. Bacterial.
84,
I., AND GROSSOWICZ, N., Proc. Sot. Biol. Med. 96, 108 (1957). 5. O’LEARY, W. M., PANOS, C., AND HELZ, G. E., J. Bacterial. 72, 673 (1956). 6. O’LEARY, W. M., J. BacterioE. 84, 967 (1962). 7. KANT-OR, F. S., AND COLE, R. M., J. Exptl. Med. 112, 77 (1960). 8. SLADE, H. D., KNOX, G. A., AND SLAMP, W. C., J. Bacterial. 62, 669 (1951). 9. SMITH, P. F., AND BOUGHTON, J. E., J. Bacteriol. 80, 851 (1960). 4.
GINSBURG,
Exptl.