Chemical Composition of the Washed Cells of Streptococcus sanguis (804) and Streptococcus mutans (B-14) M. L. SHARMA and E. NEWBRUN Section of Biological Sciences, Division of Oral Biology, School of Dentistry, University of California, San Francisco, California 94113, USA
Washed cells of Streptococcus sanguis (804) and Streptococcus mutans (B-14) were analyzed for water, ash, protein, carbohydrate, lipid, and nucleic acid content. Lipids were analyzed by gas chromatography. Ash was subjected to elemental analysis. These data were compared with the'published values for the composition of dental plaque. Dental plaque is widely considered to be a key factor in dentogingival pathology. Microorganisms belonging to the groups Streptococcus sanguis and Streptococcus mutans are the predominant streptococcal strains of plaque.' There have been numerous studies on the chemical composition of dental plaque.2-8 In particular, the carbohydrate components have been investigated because of their dual role in facilitating cell adherence and in serving as transient reservoirs of fermentable carbohydrates.9-12 There is little information on the chemical composition of the microorganisms alone which consititute about 70% of the dental plaque.13 The purpose of this study is to determine the chemical composition of the washed cells of S sanguis (804) and S mutans (B-14) grown on a dialyzed medium supplemented with glucose. For comparison and control, cells of Escherichia coli and media were subjected to similar analytical procedures. The analyses included water, ash, lipid, carbohydrate, protein, and nucleic acid content of the cells. The lipid extracts of the cells were analyzed for the component fatty acids. Ash obtained from the cells and This work was supported by Grant DE 03108 from the National Institute of Dental Research, National Institutes of Health, Bethesda, Md. Received for publication June 6, 1974. Accepted for publication October 10, 1974.
the media used for growing the cultures were subjected to elemental analysis. Materials and Methods A fermentora equipped with automatic pH controller was used for growing the cultures. Proteins, carbohydrates, and nucleic acids were determined spectrophotometrically.b Cells were fragmented by sonic disruption.c For determination of inorganic constituents, cells were incineratedd to ash at 650 C and analyzed by flame emission spectroscopy. The chemicals and reagents used-such as diphenylamine,e orcinol,e deoxyribonucleicf and ribonucleic acidf-were of analytical grade. Trypone,9 tryptose,g yeast extract,g and casamino acidsg were used for preparing the culture media. ISOLATION OF THE CELLS.-An inoculum (50 ml) of S sanguis, strain 80414 grown for 12 hours in dialyzed medium'5 was added to 9 liters of the same medium supplemented with 1% (w/v) glucose and maintained at 37 C. The culture started growing in about four hours and attained the stationary phase after 13 hours, as marked by a constant value of the absorbance at 420 nm and also by a decline in acid formation. At this stage, the cells were harvested by the continuous centrifugation at 12,100 X g and washed with 0.15 M NaCl until free of culture medium. Cellular dry weight was determined a Microferm, New Brunswick Scientific Co., New Brunswick, NJ. b Model DB-G spectrophotometer, Beckman Instruments, Inc., Fullerton, Calif. c Biosonic Model IV Sonicator, Melrose Park, Ill. d Blue M Mufflle Furnace, Blue M Electric Co., Blue Island, Ill. e Sigma Chemical Co., St. Louis, Mo. f Calbiochem, Los Angeles, Calif. g Difco Laboratories, Detroit, Mich.
482 Downloaded from jdr.sagepub.com at MOUNT ALLISON UNIV on June 12, 2015 For personal use only. No other uses without permission.
S SANGUIS
Vol 54 No. 3
lyophilized preparations. A culture of S was grown and harvested as described. The medium for growing E coli strain HB 134 consisted of 11 gm Trypone, 22.5 gm yeast extract, 4 gm glycerol made to 1 liter to which was added 67 ml of 1 M potassium phosphate buffer (pH, 7.0). ASH DETERMINATION.-A sample of the dried cell preparation (about 300 mg) was heated at 650 C for 16 hours in a tared platinum crucible. These conditions yielded a constant weight of ash. LIPID CONTENT.-Dried cells (1.5 gm) were extracted in Soxhlet apparatus for 36 hours with 300 ml of chloroform:methanol (2:1, v/v) containing a few drops of hydroquinone solution (0.1% in ethanol). The solvent was flash-evaporated in vacuo and the residue was weighed. A small portion of the lipid extract was saponified with saturated KOH in ethanolwater (1:1). The unsaponified material was extracted with petroleum ether (40 to 60 C) and discarded. The aqueous phase containing the saponified fats was acidified with sulfuric acid and extracted with petroleum ether. After evaporating the solvent, the fatty acids were esterified by refluxing in acidic methanol (sulfuric acid) for about 30 minutes. The methyl esters were identified by gas chromatography using a flame ionization detector.h The stainless steel mutans (B-14)
was
i Pentex, Mills Laboratories Inc., Kankakee, Ill.
Varian 2100, Walnut Creek, Calif.
TABLE 1 CHEMICAL COMPOSITION OF S SANGUIS, S MUTANS, E COLI, Component
S sanguis
S mutans
%
Asht
84.2 `1.4 7.0±+ 0.2
Proteint
20.8 ± 1.7 19.2 ± 2.2
Watert
Organict
Carbohydratet
Lipidl
RNA§ DNA§
8.8
483
column, 6 feet long and 2.4 mm in diameter packed with 16% of diethylene glycolsuccinate polyester in silanized Chromosorb-W, diatomaceous earth specially prepared as a solid support for GLC,h (60 to 80 mesh). A sample of C17 fatty acid (methyl ester) was incorporated as an internal standard to assign the relative values of retention times to corresponding fatty acids. PROTEIN CONTENT.-Dried cells were accurately weighed (between 24 to 26 mg), stirred in 10 ml of 2 N NaOH for two hours at room temperature and then centrifuged at 12,100 X g for ten minutes. A portion (2.5 ml) of the supernatant fluid was diluted to 25 ml with distilled water. Aliquots from this solution were analyzed for protein16 using bovine serum albumini as a standard. CARBOHYDRATE CONTENT.-A sample of dried cells (about 25 mg) was stirred in 5.0 ml of 90% (v/v) sulfuric acid for about four hours and the suspension centrifuged at 12,100 X g for ten minutes. Small aliquots of the supernatant fluid were analyzed for total carbohydrate content by the Dubois phenolsulfuric acid method,17 using glucose as a standard. Error from absorption at 490 nm was minimized by a 50- to 70-fold dilution of the solutions. NUCLEIC ACID CONTENT.-A sample (about 500 mg) of the dry cells was stirred for
on
h
S MUTANS COMPOSITION
Wet
AND
E coli
DENTAL PLAQUE Plaque
Weight (Mean-±SD)
86.5 ±+1.1 6.9 ± 0.3
7.0
71.0 +±0-9 8.2 + 0.4
78-80 10
20.8
% Dry Weight (Mean ± SD) 40-50 20.4 2.0 42.7 ±2.3 3.3 ± 0.9 13-17 12.7 ± 1.5 10-14 4.3 0.5 21.7 2.1 5.4 0.4 6.3 0.4... 4.2 ±0.4 2.4+ 0.4 1.6 0.2 1.1 +±0.2 2.9 0.2
0 See reference 7. t Mean of four determinations. $ Mean of three determinations. § Mean of two determinations.
Downloaded from jdr.sagepub.com at MOUNT ALLISON UNIV on June 12, 2015 For personal use only. No other uses without permission.
484
J Dent Res May-June 1975
SHARMA AND NEWBRUN
about 12 hours in 10 ml of 0.1 M sodium citrate in 0.01 M NaCl. This suspension was immersed in an ice bath and sonicated at 10 kc/sec at 50% output for 15-second intervals for a total of three minutes. This duration of sonication was found optimal, since prolonged duration of sonication did not release any further amounts of nucleic acids. The nucleic acids were isolated and the non-nucleic acid components were removed by precipitation with sodium lauryl sulfate.18 Traces of proteins were removed by phenol extraction. The final volume of the aqueous phase containing the nucleic acids was adjusted to 50 ml with water. This solution gave an ultraviolet absorption with a Xmax at 260 nm. Suitable aliquots were analyzed for deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) content by the diphenylamine19 and orcinol20 methods, respectively. Results CONTENT.-The water and ash content of the bacterial cells are presented in Table 1. The -water content of the two streptococcal strains were similar, and higher than that of E coli. Ash contents of the three strains were similar. The organic content, calculated by determining the difference between dry weight and ashed weight, was highest for E coli. A spectrochemical elemental analysis of the ash of the streptococci revealed that the two strains were quite similar in their inorganic composition but a high content of calcium (Ca), magnesium (Mg), and silicon (Si), and lower content of sodium (Na), were observed in the ash from E coli (Table 2). Ash from cytoplasm of S sanguis showed WATER
AND ASH
a higher content of Mg, as compared to whole cells. This may be attributed to coenzymic role of Mg in the intracellular phase. Generally, the elemental composition of the cells paralleled that of the culture medium with the exception of potassium which was excluded, iron which was concentrated by the streptococci, and silica which was accumulated by E coli cells. ORGANIC CONTENT.-Analytical data on protein, carbohydrate, lipid, and nucleic acid content of the three strains are given in Table 1. Streptococcal strains had a lower content of proteins and lipids and a higher content of carbohydrate than E coli. S sanguis had about 1.5 times the carbohydrate content of S mutans. The RNA and DNA content of the three strains are given in Table 1. The distribution of the various fatty acids from the lipid fraction and their amounts are summarized in Table 3. In general, E coli showed a higher proportion of unsaturated fatty acids than the streptococcal
strains.
Discussion Since E coli has been extensively analyzed, it was used as a control for the analytical procedures that were used. Some of the differences in protein, carbohydrate, and lipid content from published data2l may be due to strain differences and growth stage at which the culture was harvested. The cells of streptococcal strains contained a higher water and carbohydrate (Table 1) content than those of E coli. A markedly higher lipid content of E colt (fourfold to fivefold) is characteristic of gram-negative strains.22 This difference in lipid content can be used as a differentiating characteristic. Analysis of
TABLE 2 ELEMENTAL ANALYSES OF THE ASH OBTAINED FROM VARIOUS STRAINS OF CELLS AND THE MEDIA USED FOR THEIR GROWTH Dialyzed Elem-ent
B Ca Fe K
Mg Mn Na
P Si Zn
S mutans
Medium(%) (B-14) (0% )
0.05 0.30
Washed cells of Streptococcus sanguis (804) and Streptococcus mutans (B-14) were analyzed for water, ash, protein, carbohydrate, lipid, and nucleic acid content. Lipids were analyzed by gas chromatography. Ash was subjected to elemental analysis. These data were compared with the'published values for the composition of dental plaque. Dental plaque is widely considered to be a key factor in dentogingival pathology. Microorganisms belonging to the groups Streptococcus sanguis and Streptococcus mutans are the predominant streptococcal strains of plaque.' There have been numerous studies on the chemical composition of dental plaque.2-8 In particular, the carbohydrate components have been investigated because of their dual role in facilitating cell adherence and in serving as transient reservoirs of fermentable carbohydrates.9-12 There is little information on the chemical composition of the microorganisms alone which consititute about 70% of the dental plaque.13 The purpose of this study is to determine the chemical composition of the washed cells of S sanguis (804) and S mutans (B-14) grown on a dialyzed medium supplemented with glucose. For comparison and control, cells of Escherichia coli and media were subjected to similar analytical procedures. The analyses included water, ash, lipid, carbohydrate, protein, and nucleic acid content of the cells. The lipid extracts of the cells were analyzed for the component fatty acids. Ash obtained from the cells and This work was supported by Grant DE 03108 from the National Institute of Dental Research, National Institutes of Health, Bethesda, Md. Received for publication June 6, 1974. Accepted for publication October 10, 1974.
the media used for growing the cultures were subjected to elemental analysis. Materials and Methods A fermentora equipped with automatic pH controller was used for growing the cultures. Proteins, carbohydrates, and nucleic acids were determined spectrophotometrically.b Cells were fragmented by sonic disruption.c For determination of inorganic constituents, cells were incineratedd to ash at 650 C and analyzed by flame emission spectroscopy. The chemicals and reagents used-such as diphenylamine,e orcinol,e deoxyribonucleicf and ribonucleic acidf-were of analytical grade. Trypone,9 tryptose,g yeast extract,g and casamino acidsg were used for preparing the culture media. ISOLATION OF THE CELLS.-An inoculum (50 ml) of S sanguis, strain 80414 grown for 12 hours in dialyzed medium'5 was added to 9 liters of the same medium supplemented with 1% (w/v) glucose and maintained at 37 C. The culture started growing in about four hours and attained the stationary phase after 13 hours, as marked by a constant value of the absorbance at 420 nm and also by a decline in acid formation. At this stage, the cells were harvested by the continuous centrifugation at 12,100 X g and washed with 0.15 M NaCl until free of culture medium. Cellular dry weight was determined a Microferm, New Brunswick Scientific Co., New Brunswick, NJ. b Model DB-G spectrophotometer, Beckman Instruments, Inc., Fullerton, Calif. c Biosonic Model IV Sonicator, Melrose Park, Ill. d Blue M Mufflle Furnace, Blue M Electric Co., Blue Island, Ill. e Sigma Chemical Co., St. Louis, Mo. f Calbiochem, Los Angeles, Calif. g Difco Laboratories, Detroit, Mich.
482 Downloaded from jdr.sagepub.com at MOUNT ALLISON UNIV on June 12, 2015 For personal use only. No other uses without permission.
S SANGUIS
Vol 54 No. 3
lyophilized preparations. A culture of S was grown and harvested as described. The medium for growing E coli strain HB 134 consisted of 11 gm Trypone, 22.5 gm yeast extract, 4 gm glycerol made to 1 liter to which was added 67 ml of 1 M potassium phosphate buffer (pH, 7.0). ASH DETERMINATION.-A sample of the dried cell preparation (about 300 mg) was heated at 650 C for 16 hours in a tared platinum crucible. These conditions yielded a constant weight of ash. LIPID CONTENT.-Dried cells (1.5 gm) were extracted in Soxhlet apparatus for 36 hours with 300 ml of chloroform:methanol (2:1, v/v) containing a few drops of hydroquinone solution (0.1% in ethanol). The solvent was flash-evaporated in vacuo and the residue was weighed. A small portion of the lipid extract was saponified with saturated KOH in ethanolwater (1:1). The unsaponified material was extracted with petroleum ether (40 to 60 C) and discarded. The aqueous phase containing the saponified fats was acidified with sulfuric acid and extracted with petroleum ether. After evaporating the solvent, the fatty acids were esterified by refluxing in acidic methanol (sulfuric acid) for about 30 minutes. The methyl esters were identified by gas chromatography using a flame ionization detector.h The stainless steel mutans (B-14)
was
i Pentex, Mills Laboratories Inc., Kankakee, Ill.
Varian 2100, Walnut Creek, Calif.
TABLE 1 CHEMICAL COMPOSITION OF S SANGUIS, S MUTANS, E COLI, Component
S sanguis
S mutans
%
Asht
84.2 `1.4 7.0±+ 0.2
Proteint
20.8 ± 1.7 19.2 ± 2.2
Watert
Organict
Carbohydratet
Lipidl
RNA§ DNA§
8.8
483
column, 6 feet long and 2.4 mm in diameter packed with 16% of diethylene glycolsuccinate polyester in silanized Chromosorb-W, diatomaceous earth specially prepared as a solid support for GLC,h (60 to 80 mesh). A sample of C17 fatty acid (methyl ester) was incorporated as an internal standard to assign the relative values of retention times to corresponding fatty acids. PROTEIN CONTENT.-Dried cells were accurately weighed (between 24 to 26 mg), stirred in 10 ml of 2 N NaOH for two hours at room temperature and then centrifuged at 12,100 X g for ten minutes. A portion (2.5 ml) of the supernatant fluid was diluted to 25 ml with distilled water. Aliquots from this solution were analyzed for protein16 using bovine serum albumini as a standard. CARBOHYDRATE CONTENT.-A sample of dried cells (about 25 mg) was stirred in 5.0 ml of 90% (v/v) sulfuric acid for about four hours and the suspension centrifuged at 12,100 X g for ten minutes. Small aliquots of the supernatant fluid were analyzed for total carbohydrate content by the Dubois phenolsulfuric acid method,17 using glucose as a standard. Error from absorption at 490 nm was minimized by a 50- to 70-fold dilution of the solutions. NUCLEIC ACID CONTENT.-A sample (about 500 mg) of the dry cells was stirred for
on
h
S MUTANS COMPOSITION
Wet
AND
E coli
DENTAL PLAQUE Plaque
Weight (Mean-±SD)
86.5 ±+1.1 6.9 ± 0.3
7.0
71.0 +±0-9 8.2 + 0.4
78-80 10
20.8
% Dry Weight (Mean ± SD) 40-50 20.4 2.0 42.7 ±2.3 3.3 ± 0.9 13-17 12.7 ± 1.5 10-14 4.3 0.5 21.7 2.1 5.4 0.4 6.3 0.4... 4.2 ±0.4 2.4+ 0.4 1.6 0.2 1.1 +±0.2 2.9 0.2
0 See reference 7. t Mean of four determinations. $ Mean of three determinations. § Mean of two determinations.
Downloaded from jdr.sagepub.com at MOUNT ALLISON UNIV on June 12, 2015 For personal use only. No other uses without permission.
484
J Dent Res May-June 1975
SHARMA AND NEWBRUN
about 12 hours in 10 ml of 0.1 M sodium citrate in 0.01 M NaCl. This suspension was immersed in an ice bath and sonicated at 10 kc/sec at 50% output for 15-second intervals for a total of three minutes. This duration of sonication was found optimal, since prolonged duration of sonication did not release any further amounts of nucleic acids. The nucleic acids were isolated and the non-nucleic acid components were removed by precipitation with sodium lauryl sulfate.18 Traces of proteins were removed by phenol extraction. The final volume of the aqueous phase containing the nucleic acids was adjusted to 50 ml with water. This solution gave an ultraviolet absorption with a Xmax at 260 nm. Suitable aliquots were analyzed for deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) content by the diphenylamine19 and orcinol20 methods, respectively. Results CONTENT.-The water and ash content of the bacterial cells are presented in Table 1. The -water content of the two streptococcal strains were similar, and higher than that of E coli. Ash contents of the three strains were similar. The organic content, calculated by determining the difference between dry weight and ashed weight, was highest for E coli. A spectrochemical elemental analysis of the ash of the streptococci revealed that the two strains were quite similar in their inorganic composition but a high content of calcium (Ca), magnesium (Mg), and silicon (Si), and lower content of sodium (Na), were observed in the ash from E coli (Table 2). Ash from cytoplasm of S sanguis showed WATER
AND ASH
a higher content of Mg, as compared to whole cells. This may be attributed to coenzymic role of Mg in the intracellular phase. Generally, the elemental composition of the cells paralleled that of the culture medium with the exception of potassium which was excluded, iron which was concentrated by the streptococci, and silica which was accumulated by E coli cells. ORGANIC CONTENT.-Analytical data on protein, carbohydrate, lipid, and nucleic acid content of the three strains are given in Table 1. Streptococcal strains had a lower content of proteins and lipids and a higher content of carbohydrate than E coli. S sanguis had about 1.5 times the carbohydrate content of S mutans. The RNA and DNA content of the three strains are given in Table 1. The distribution of the various fatty acids from the lipid fraction and their amounts are summarized in Table 3. In general, E coli showed a higher proportion of unsaturated fatty acids than the streptococcal
strains.
Discussion Since E coli has been extensively analyzed, it was used as a control for the analytical procedures that were used. Some of the differences in protein, carbohydrate, and lipid content from published data2l may be due to strain differences and growth stage at which the culture was harvested. The cells of streptococcal strains contained a higher water and carbohydrate (Table 1) content than those of E coli. A markedly higher lipid content of E colt (fourfold to fivefold) is characteristic of gram-negative strains.22 This difference in lipid content can be used as a differentiating characteristic. Analysis of
TABLE 2 ELEMENTAL ANALYSES OF THE ASH OBTAINED FROM VARIOUS STRAINS OF CELLS AND THE MEDIA USED FOR THEIR GROWTH Dialyzed Elem-ent
B Ca Fe K
Mg Mn Na
P Si Zn
S mutans
Medium(%) (B-14) (0% )
0.05 0.30
