JOURNAL
OF
BACTERIOLOGY, Jan. 1977,
p.
Vol. 129, No. 1 Printed in U.S.A.
445-456
Copyright X) 1977 American Society for Microbiology
Ultrastructural, Physiological, and Cytochemical Characterization of Cores in Group D Streptococcil SYLVIA E. COLEMAN2* AND ARNOLD S. BLEIWEIS Department of Microbiology and Cell Science, University ofFlorida, Gainesville, Florida 32611 Received for publication 3 August 1976
Cores are large, rod-shaped structures that have been found almost exclusively in group D streptococci, measure 0.1 to 0.16 ,um in diameter, and extend the width or length of cells. This study has shown that cores are produced in the cells at a reproducible point in early stationary growth after extensive mesosomal formation and after the pH has dropped below 6.5. When cells containing cores were introduced into a fresh medium with a pH above 6.5, the structures disappeared within 5 min. The structures were not found in young, logarithmically growing cells but formed in these cells upon autolysis or treatment with penicillin. Cores that were forming or disintegrating appeared to have a lamellar substructure. When chloramphenicol was added to the medium before the culture reached stationary phase, no cores were found in the cells. Cytochemical studies indicated that cores contain protein and are not composed of cell wall material or other polysaccharides that contain 1,2-glycol groups.
Cores are large, hollow, cylindrical structures that have been found almost exclusively in group D streptococci (16, 17) and were first observed by Abrams et al. (1) in 1964. These structures extend the width or length of a dividing cell and measure 0.1 to 0.16 ,um in diameter, with a tube wall that is between 0.01 and 0.03 ,um thick (3, 17). Cores normally form in stationary-phase cultures and have been observed after treatment with penicillin (R. G. McCandless, M. Cohen, G. M. Kalmanson, and L. B. Guze, J. Ultrastruct. Res. 21:162-163, 1967). Little is known of the chemical or physiological properties of these unusual structures. The work reported here shows that cores: (i) form at a reproducible point in early stationary growth phase after a period of extensive mesosomal formation; (ii) form upon autolysis or upon treatment with penicillin; (iii) are composed of protein and probably little or no carbohydrate; (iv) form in a growing culture only after the pH drops below 6.5; (v) do not form in the presence of chloramphenicol (CAP); and (vi) possess a lamellar substructure observable in cores that appear to be forming or disintegrating. The function of these structures has not been defined, but it is postulated that they may repre-
sent a labile repository for cellular protein, perhaps membranous in origin.
I Authorized for publication as paper no. 6174 in the Journal Series of the Florida Agricultural Experiment Station. 2 Present address: Research Service 151, Veterans Administration Hospital, Gainesville, FL 32602.
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MATERIALS AND METHODS Cultures and media. Two strains of group D streptococci were used: Streptococcus faecalis ATCC 8043 and S. faecalis XL, a laboratory isolate. The bacteria were cultured in Todd-Hewitt broth (Difco), adjusted to a final pH of 7.2, and on agar plates of Todd-Hewitt broth with 2% agar added. Balanced cultural growth. Cultures were grown under conditions in which absorbance and dry weight were increasing at the same rate, and the lag phase was eliminated. Initial inocula of 0.1 ml of an overnight broth culture were spread on plates of Todd-Hewitt agar, which were incubated at 37°C overnight. Washings from a plate were inoculated into a flask containing 100 ml of broth prewarmed to 37°C and incubated to mid-logarithmic growth phase. From this culture, 30 ml was inoculated into a second prewarmed flask of 300 ml, and sequential inocula of 30 ml taken from mid-logarithmic phase were put into 300 ml of broth until balanced cultural growth was obtained. Dry weights were determined by filtering measured amounts of broth through a Nuclepore filter (General Electric Co.) with a pore size of 0.5 ,um and drying and weighing the filters. Turbidity was measured on a Gilford spectrophotometer at 660 nm. Cytochemical determination of polysaccharides. The presence of polysaccharides containing 1,2-glycol groups was detected in thin sections by the silver methenamine method (9, 26). The bacteria were fixed with glutaraldehyde and osmium tetroxide and embedded in an Epon-Araldite plastic mixture (18), and thin sections were placed on stainless-steel
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COLEMAN AND BLEIWEIS
grids for staining. After oxidation with 1% periodic acid for 5 min at room temperature, the grids were washed and placed in the staining solution (100 ml of 3% hexamethylenetetramine [Eastman Organic Chemicals, Rochester, N.Y.] and 5 ml of 5% silver nitrate, both in double-distilled water). Enzyme digestion of fixed preparations. Bacteria were fixed in 2% glutaraldehyde in the cold and then embedded in water-soluble glycol-methacrylate (14). Thin sections were placed on stainless-steel grids and oxidized for 1 min with 3% hydrogen peroxide before exposure to the following enzymes (19,27) at 37°C: (i) Pronase (type VI, Sigma Chemical Co., St. Louis, Mo.), 0.2% in 0.2 M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride buffer (pH 7.4) for 10, 20, 30, and 45 min; and (ii) lysozyme (egg white, grade 1, Sigma Chemical Co.), 25,000 ,ug per ml in 0.01 M Tris-hydrochloride buffer (pH 8.3) for 30 min, 1, 2, and 6 h, followed by treatment with 2% sodium dodecyl sulfate for 10 min. Induction of cores by autolysis. Young cells from logarithmically growing cultures were harvested by centrifugation, washed three times with distilled water, and allowed to autolyze in 0.01 M potassium phosphate buffer (pH 6.5) at 37°C for periods of 15, 30, and 60 min (25). Cells in the process of autolysis were examined for core formation by electron microscopy. Induction of cores by penicillin. Cores were induced in cells from logarithmically growing cultures by the addition of 1,000 U of potassium penicillin G (Eli Lilly and Co., Indianapolis, Ind.) per ml for periods of 5, 10, 30, and 45 min, after which samples were fixed and embedded for observation with the electron microscope. Formation of spheroplasts. Spheroplasts of cells containing cores were prepared by the method of Coleman et al. (5, 6) by taking bacteria from stationary-phase culture and washing and incubating them for 30 min at 37°C with 250 jig of lysozyme per ml. Effect of pH on stability of cores. Cells and spheroplasts were incubated for 30 and 60 min at 37°C in the following buffers, with 0.5 M sucrose added for spheroplasts: 0.1 M HCl-KCl buffer (pH 2.0), 0.1 M citrate buffer (pH 4.0), 0.1 M potassium phosphate buffer (pH 7.0), 0.1 M Tris buffer (pH 7.2), 0.1 M potassium phosphate buffer (pH 8.0), and 0.1 M Tris buffer (pH 8.4). In experiments in which cells were grown in a buffered medium, Todd-Hewitt broth was prepared with 0.01 M potassium phosphate buffer (pH 7.2). Inhibition of core synthesis. The production of cores was inhibited by the addition of 50 Mug of CAP (Sigma Chemical Co.) per ml at 30 min or 1.5 h before formation of cores normally occurred in the culture. Electron microscopy. Various fixation techniques were used, including the method of Ryter et al. (24); various times of fixation were also used: 2% glutaraldehyde was utilized alone or followed by 1% osmium tetroxide, both in the cold and at room temperature, and sometimes preparations were postfixed with 2% uranyl acetate in buffer at room temperature. Prefixation with 0.01% osmium tetroxide, followed by fixation with glutaraldehyde
J. BACTERIOL.
and postfixation with osmium tetroxide and uranyl acetate, was also used with either sodium cacodylate or Veronal buffers. Embedding was in either an Epon-Araldite plastic mixture (18) or in Araldite (8). Thin sections were cut with a diamond knife (I. E. du Pont de Nemours & Co., Wilmington, Del.) on a Porter-Blum MT-2 ultramicrotome (Ivan Sorvall, Inc., Norwalk, Conn.), poststained with uranyl acetate and lead citrate (23), and observed with an Hitachi HUll-E electron microscope (Hitachi, Ltd., Tokyo, Japan) at 75 kV. Freeze-fracture. Fixed and unfixed bacteria were treated with 30% glycerol for 20 min, frozen in liquid Freon 22, and fractured at - 100°C on a Balzers BA 360 M freeze-etch apparatus (Balzers AG, Furstentum, Liechtenstein) using the method of Moor and Muhlethaler (20). The cells were sometimes etched for 2 or 4 min. The replicas were cleaned overnight in 40% aqueous chromium trioxide, washed several times in water, and picked up on uncoated grids.
RESULTS Structure of cores in ultrathin sections and in freeze-fractured cells. Good preservation of the core structures was obtained by fixation in glutaraldehyde alone or by various combinations of glutaraldehyde and osmium tetroxide. Figure la shows a cross section and Fig. lb a longitudinal section of cores in S. faecalis XL after fixation with glutaraldehyde in the cold followed by osmium tetroxide at room temperature. The appearance of the cores after freezefracture of either unfixed (Fig. lc) or fixed cells (Fig. ld) was similar to their appearance in thin section. Cores in S. faecalis ATCC 8043 differed from those in S. faecalis XL by being denser in cross section (Fig. 2a) and more fibrous in appearance (Fig. 2b). The structure of these cores in freeze-fractured cells (Fig. 2c and d) is again similar to their morphology in thin section. Formation of cores during balanced growth. S. faecalis XL was cultured under conditions in which the lag phase was eliminated and the absorbance and dry weight of the cells were increasing at the same rate. Under these conditions, cores appeared in the cells after 3.5h growth and formed reproducibly at this point in the growth cycle (Fig. 3). During h 1 of growth, few mesosomes were observed, but beginning at 2 h extensive mesosomal membrane was observed in the cells (Fig. 4). Numerous observations of cells in many thin sections indicated that mesosomes became noticeably smaller after the formation of cores at 3.5-h growth. Persistence of core structures and their relationship to viability of the cells. Cores and mesosomes could be observed in the cells throughout 7 days of growth in Todd-Hewitt
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FIG. 1. (a) Cross section of a core in S. faecalis XL after 20-h growth in Todd-Hewitt broth. Fixation in glutaraldehyde in the cold followed by osmium tetroxide at room temperature. Bar = 0.1 um. (b) Longitudinal section of a core in S. faecalis XL after 8-h growth in Todd-Hewitt broth and fixation in glutaraldehyde in the cold followed by osmium tetroxide at room temperature. Bar = 0.1 ,um. (c) Freeze-etched unfixed cell of S. faecalis XL after 36-h growth in Todd-Hewitt broth. Inset is a cross fracture of an unfixed core. Bars = 0.1 ,um. (d) Freeze-etched cell of S. faecalis XL fixed with glutaraldehyde after 14-h growth in Todd-Hewitt broth. Inset is a cross fracture of a glutaraldehyde-fixed core. Bars = 0.1 ,um.
448
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FIG. 2. (a) Cross section of a core in S. faecalis ATCC 8043 fixed with glutaraldehyde and osmium tetroxide after 24-h growth in Todd-Hewitt broth. Bar = 0.2 gm. (b) Longitudinal section of a core in S. faecalis ATCC 8043 fixed with glutaraldehyde and osmium tetroxide after 24-h growth in Todd-Hewitt broth. Bar = 0.2 ,um. (c) Cross section ofa core in a freeze-etched cell ofS. faecalis ATCC 8043 after 24-h growth in Todd-Hewitt broth. Bar = 0.2 ,um. (d) Longitudinal fracture of a core in a freeze-etched cell of S. faecalis ATCC 8043 after 24-h growth in Todd-Hewitt broth. Bar = 0.1 Jim.
broth at 37°C. The pH of the culture decreased to about 6.2 at the time cores formed (Fig. 3) and remained this low or lower during the 7 days of growth. The formation of cores did not
affect viability as measured in colony-forming units (CFU) and a decrease in CFU was observed only after 7 days. From a count of 8.2 x 107 CFU per ml after 30 min, there was an
CORES IN GROUP D STREPTOCOCCI
VOL. 129, 1977
increase to 1 x 109 CFU per ml after 1.5 h, which remained through 48 h. At this time the pH, which was initially 7.4, had decreased to 5.9. Approximately 90% of the cells in thin sections contained cores after 3.5-h growth. Effect of CAP on the formation of cores. Thirty minutes and 1.5 h before cores formed normally, 50 jig of CAP was added to the culture (Fig. 5). In neither case did cores form after 5-h incubation, although numerous cells 2.5 1.2
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FIG. 3. Growth of a culture of S. faecalis XL in Todd-Hewitt broth at 37°C for 168 h (7 days). Changes in absorbance and pH are shown. Cores could be observed in the cells throughout the 168 h starting at 3.5 h.
449
contained cores in control cultures. Frequent accumulations of clear material were noted in the cells treated with CAP, but no cores were formed. When cells treated with CAP were inoculated into fresh medium or into filtered, spent broth from a stationary-phase culture and incubated for 24 h, cores subsequently were produced. Conditions of formation and disaggregation of cores. Since young, logarithmically growing cells do not contain cores, it was hypothesized that the structures might disappear soon after inoculation of older cells into fresh broth. Cores, which were numerous in cells taken from a stationary-phase culture, could no longer be observed 5 min after the cells were inoculated into fresh medium at pH 7.2. Cells for the inoculum in this experiment were collected at 37°C to avoid a temperature shift that might influence the disposition of the cores. Cores, however, were found to be stable at 2°C for 60 min, indicating that they do not disintegrate in the cold. Cores also remained stable after placing the cells into fresh or filtered, spent Todd-Hewitt both at pH 6.1 (Table 1). Young cells without cores, when introduced into spent medium or fresh medium at pH 6.1, did not form cores after 3.5 h even though the cells grew slowly. In control cultures, the pH dropped to 6.2
FIG. 4. Mesosomes could be observed in almost every cell in thin sections ofS. faecalis XL after 2-h growth in Todd-Hewitt broth. Fixation by the method of Ryter et al. (24). Bar = 1.0 ,utm.
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COLEMAN AND BLEIWEIS
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A
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FIG. 5. Growth of cultures of S. faecalis XL in Todd-Hewitt broth at 37°C after addition of 50 pg of CAP per ml: (A) 1.5 h before the normal time for formation of cores; (B) 30 min before core formation occurs; and (C) a control culture. TABLE 1. Changes in cells of Streptococcus faecalis XL with and without cores after inoculation into fresh and spent Todd-Hewitt broth at 37°C Cells With cores
Broth Fresh medium, pH 7.2
With cores
Fresh medium, pH 6.1
With cores
Spent broth, pH 6.1
Without cores Without cores
Spent broth, pH 6.1 Fresh medium, pH 6.1
Cores Disappear within 5 min Remain stable in the cells Remain stable in the cells No cores formed within 3 h No cores formed within 3 h
decreased to 6.2, the stability of cores in cells of S. faecalis ATCC 8043 was tested in relation to pH. Cores in cells of S. faecalis ATCC 8043 were stable after incubation for 60 min at 37°C in 0.1 M HCl-KCl buffer (pH 2.0), even though much of the cytoplasm was lost (Fig. 7). Cells incubated for 30 min at 37°C in 0.1 M citrate buffer (pH 4.0) also contained stable cores. Cores were also clearly discernible in cells placed in distilled water at pH 6.0. However, when the pH of the culture medium was raised from 6.1 to 7.0 after cores had been produced, and the culture was incubated for 30 min at 37°C, cores were no longer visible. Cells with cores that were incubated for 30 min at 37°C in 0.1 M Tris buffer (pH 7.2) no longer contained cores, nor did cells after 30 min at 37°C in 0.1 M potassium phosphate buffer (pH 8.0), nor after raising the pH of the growth medium to 8.0 for 30 min at 37°C. When the core-containing cells were chilled to 2°C for 30 min after raising the pH of the medium to 8.0, the core structures also disappeared, indicating that they were disrupted at the nonoptimal pH whether the cells were at growth temperature or in the cold. Even after a brief washing of 5 min in 0.1 M Tris buffer (pH 8.2), the cores were barely discernible. The cells remained viable after these treatments. Formation of cores in autolyzing cells and in the presence of penicillin. Young cells from
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after 2 h and was at this level at 3.5 h when cores were formed initially. In a culture of S. faecalis ATCC 8043 buffered in Todd-Hewitt broth with 0.01 M potassium phosphate buffer (pH 7.4) and incubated at 37°C, the growth was slower than normal, but after 48 h there was still no production of cores at a final pH of 6.6. Two cultures of S. faecalis XL buffered with 0.01 M citrate buffer (pH 4.5) and 0.1 M Tris buffer (pH 7.9) (Fig. 6) did not produce cells that formed cores: the former had diminished growth, and the pH of the latter only decreased to 6.5, which was apparently not low enough for the formation of cores. Effect of pH on the stability of cores. Since cores did not appear in culture until the pH
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FIG. 6. Growth of cultures of S. faecalis XL in: (A) unbuffered Todd-Hewitt broth; (B) Todd-Hewitt broth buffered with 0.01 M citrate buffer, pH 4.5; and (C) Todd-Hewitt broth buffered with 0.01 M Tris buffer, pH 7.9. Cultures were incubated at 37°C.
CORES IN GROUP D STREPTOCOCCI
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logarithmically growing cultures never contained cores. However, when these cells were permitted to autolyze in 0.1 M potassium phosphate buffer (pH 6.5) at 37°C, cores were formed as autolysis proceeded. Autolysis began in the septal area (11) and, after 15 to 30 min, large accumulations of granular material formed in the septal area of cells of S. faecalis ATCC 8043 (Fig. 8a). The structure of the cores that formed beginning at 30-min autolysis can be seen in S. faecalis ATCC 8043 (Fig. 8b). After 60-min autolysis, the cells began to lyse. Cores also could be induced in young cells by adding 1,000 U of penicillin G per ml to cultures at mid-logarithmic growth and incubating them an additional 30 min. Structure of cores in spheroplasts. The treatment of core-containing cells of S. faecalis XL with 250 ,ug of lysozyme per ml, at 37°C in distilled water at pH 6.0 for the formation of spheroplasts, resulted in the apparent disintegration of the core structure after only 5-min treatment with lysozyme. The cores degraded into layers of dense material (Fig. 9a and b). The layered substructure in the cores of spheroplasts was also evident in freeze-fractured preparations of unfixed spheroplasts (Fig. 9c and d). Intermediate forms of cores. Occasional forms could be seen in thin section (Fig. 10a) and in freeze-fracture (Fig. 10b) of older cells that appeared to be intermediates of cores, sim-
ilar to those seen in penicillin-induced spheroplasts. Cytochemical and enzymatic studies of core structure. To determine the possible presence in the core structures of polysaccharides containing 1,2-glycol groups, the silver methenamine staining procedure was used. Although the cell walls stained heavily (Fig. Ila and b), the core structures did not contain polysaccharides of these specifications. Cores disappeared from thin sections of cells embedded in glycol-methacrylate after digestion with Pronase for 45 min (Fig. 12a). This suggests the presence of a protein component in the cores. Digestion of sections with 25,000 ,ug of lysozyme per ml (Fig. 12b) removed the cell wall but not the core structure. This suggests that substantial amounts of peptidoglycan are not present in the core. Digestion of cores was also attempted with 0.1 % ribonuclease, deoxyribonuclease, lipase, and 0.5% trypsin with inconsistent or negative results.
DISCUSSION The fact that the structure of the core was the same in fixed, embedded cells and in freezefractured, unfixed cells established a basis for further work on the properties, development, and stability of these unusual structures. The
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FIG. 8. (a) Section showing accumulations of material near septal area of S. faecalis ATCC 8043 after 30min autolysis in 0.01 M phosphate buffer, pH 6.5. Bar = 0.1 um. (b) Thin section ofa core forming in a cell of S. faecalis ATCC 8043 after autolysis for 30 min at 37°C in 0.01 Mphosphate buffer, pH 6.5. Bar = 0.1 ,um.
main chemical and physical characteristics of and Smith (7) postulated that the microtubular cores are their lability at higher pH and the structures that they observed in L-forms of S. apparent protein nature of the structures. Dis- faecalis var. zymogenes could be aberrant cernible subunits were not observed in the forms of the normal mesosome that were no cores, although most other tubular structures longer observed in bacterial protoplasts and Lin prokaryotic cells, such as gas vacuoles (2, 4), forms. It may be that mesosomal membrane in spore appendages (22, 29), and rhapidosomes (15, 28), have a regular arrangement of sub- group D streptococci has a structural specificity units. Layers of substructural material could be that causes it to disassemble and reassemble in observed both in cores forming in autolyzing a tubular configuration. The mesosomes in S. cells (Fig. 8b) and in cores that were apparently faecalis (10) occur in a baglike shape that could disintegrating in spheroplasts (Fig. 9a and b). be visualized as elongating into a tube (Fig. lOa The layers of protein-containing substructural and b). The problem involved in recognizing an components are apparently formed only when intermediate stage with certainty makes this the pH has decreased to 6.2, indicating that the difficult to investigate. protein is in an altered form at higher pH. It Higgins and Daneo-Moore (10) suggested also seems from the results of polysaccharide that the formation of mesosomal membrane in staining (Fig. lla and b) that cores do not con- S. faecalis ATCC 9790 depends upon the rate of tain cell wall components, as was suggested synthesis of deoxyribonucleic acid. During acearlier by Cohen et al. (3). tive, logarithmic growth, large amounts of mesSeveral workers have observed rodlike struc- osomal membrane are produced, as was obtures to which they attributed a membranous served in S. faecalis XL. When the cultures origin in streptococci and other bacteria. Kuhrt reached stationary growth phase, the rate of and Pate (Bacteriol. Proc., p. 165, 1971) ob- deoxyribonucleic acid synthesis slowed. It is served large amounts of mesosomal membrane shortly after this period that smaller mesosoin cells of Chondrococcus columnaris which, mal structures are seen (10). upon lysis, released large numbers of small Apparently, the specific protein(s) that goes microtubular structures which they concluded, into the core is present in the cells during logafrom a comparison of the chemical composi- rithmic growth but is arranged into a core only tions, were derived from the mesosomal mem- when growth is interrupted. This rearrangebrane. Studying group D streptococci, Corfield ment to form a core does not occur when protein
VOL. 129, 1977
CORES IN GROUP D STREPTOCOCCI
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FIG. 9. (a) Cross section of an apparently disintegrating core in a spheroplast of S. faecalis XL after exposure to 250 pg of lysozyme per ml for 30 min at 37°C in distilled water + 1% glucose at pH 6.0. Bar = 0.2 p.m. (b) Longitudinal section showing filamentous structure of an apparently disintegrating core in a spheroplast of S. faecalis XL after exposure to 250 pg of lysozyme per ml for 30 min at 37°C in distilled water + 1% glucose, pH 6.0. Bar = 0.2 pm. (c) Freeze-etched preparation showing a longitudinal fracture of a disintegrating core in a spheroplast ofS. faecalis XL after exposure to 250 pg of lysozyme per ml for 30 min at 37°C in the presence of 0.5 M sucrose. Bar = 0.2 ,um. (d) Cross fracture of a core in a freeze-etched spheroplast of S. faecalis XL after exposure to 250 pg of lysozyme per ml for 30 min in 0.5 M sucrose. Bar = 0.2 ,um.
synthesis is inhibited by CAP, indicating that some factor involved in the formation of cores must be affected by CAP. There are several possible explanations for this. First, perhaps
there is an enzyme(s) or structural protein that must be produced de novo as the core is formed. Second, mesosomal membrane is rarely seen in cells treated with CAP, and cores would not be
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9
FIG. 10. (a) Section ofa possible intermediate form of a core in S. faecalis ATCC 8043. Bar = 0.2 um. (b) Freeze-etched cell of S. faecalis XL showing a possible intermediate form of a core. Bar = 0.2 p.tm. _t
FIG. 11. (a) Silver methenamine stain of a section of S. faecalis XL showing heavy deposition of stain in the cell wall and none in the longitudinal core structure. Bar = 0.1 pm. (b) Cross section of a core unstained by the silver methenamine method with heavy staining of the cell wall polysaccharide. Bar = 0.2 pum.
formed if they were derived from mesosomal membrane whose synthesis was inhibited by CAP. The third possibility is derived from the work of Pooley and Shockman (21), who found
over an 80% decrease in autolytic activity of cells of S. faecalis ATCC 9790 within 10 min after treatment of cells with 100 ug of CAP per ml. If cellular autolysis is involved in the pro-
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FIG. 12. (a) Section of glutaraldehyde-ftxed S. faecalis XL showing digestion of the core structure after treatment of the section with 0.2% Pronase in 0.2 M Tris-hydrochloride buffer (pH 7.4) for 45 min at 37°C. Bar = 0.2 pum. (b) Section of glutaraldehyde-fixed S. faecalis XL showing digestion of the cell wall but no effect on the core structure after exposure to 25,000 pg of lysozyme per ml in 0.01 M Tris-hydrochloride buffer (pH 8.3) for 60 min at 37°C. Bar = 0.2 pum.
duction of cores, then CAP could be inhibiting the process by interfering with autolysis. Recent studies with S. faecalis have revealed that lipoteichoic acid (LTA) is secreted into the medium during formation of osmotically fragile bodies from exponential-phase cells (13). Evidence was obtained that the LTA was not associated with isolated mesosomal vesicles. In Staphylococcus aureus, however, Huff et al. (12) showed that mesosomal vesicles were the major location of LTA. In relation to core structures, it seems unlikely that the group D antigen, which has a-1, 2-linked glucose units (kojibiose) as the immunodeterminant group, would be present in any substantial quantity because of the failure of cores to stain by the silver methenamine method. However, this does not rule out some involvement (16) of LTA in the process of core formation. Immunocytochemical studies currently are in progress to verify the cellular localization of LTA in membranous structures of S. faecalis. ACKNOWLEDGMENTS We wish to thank H. C. Aldrich for his advice throughout this study and for the use of the facilities of the Ultrastructure Laboratory in the Department of Botany. We thank E. P. Previc for his critical reading of the manuscript and J. Duggan for help with the figures. This research was supported by grants from the Florida Agricultural Experiment Station (BC-01440) and a Public Health Service grant from the National Institute of Dental Research (DE-02901-08).
LITERATURE CITED 1. Abrams, A., L. Nielsen, and J. Thaemert. 1964. Rapidly synthesized ribonucleic acid in membrane ghosts from Streptococcus faecalis protoplasts. Biochim. Biophys. Acta 80:325-337. 2. Bowen, C. C., and T. E. Jensen. 1965. Blue-green algae: fine structure of the gas vacuoles. Science 147:14601462. 3. Cohen, M., R. G. McCandless, G. M. Kalmanson, and L. B. Guze. 1968. Core-like structures in transitional and protoplast forms of Streptococcus faecalis, p. 94109. In L. B. Guze (ed.), Microbial protoplasts, spheroplasts, and L-forms. The Williams & Wilkins Co., Baltimore. 4. Cohen-Bazire, G., R. Kunisawa, and N. Pfennig. 1969. Comparative study of the structure of gas vacuoles. J. Bacteriol. 100:1049-1061. 5. Coleman, S. E., I. van de Rijn, and A. S. Bleiweis. 1970. Lysis of grouped and ungrouped streptococci by lysozyme. Infect. Immun. 2:563-569. 6. Coleman, S. E., I. van de Rin, and A. S. Bleiweis. 1971. Lysis of cariogenic and noncariogenic oral streptococci with lysozyme. J. Dent. Res. 50:939-943. 7. Corfield, P. S., and D. G. Smith. 1968. Microtubular structures in group D streptococcal L-forms. Arch. Mikrobiol. 63:356-361. 8. Coulter, H. D. 1967. Rapid and improved methods for embedding biological tissues in Epon 812 and Araldite 502. J. Ultrastruct. Res. 20:346-355. 9. deMartino, C., and L. Zamboni. 1967. Silver methenamine stain for electron microscopy. J. Ultrastruct. Res. 19:273-282. 10. Higgins, M. L., and L. Daneo-Moore. 1972. Morphokinetic reactions of cells of Streptococcus faecalis (ATCC 9790) to specific inhibition of macromolecular synthesis: dependence of mesosome growth on deoxyribonucleic acid synthesis. J. Bacteriol. 109:12211231. 11. Higgins, M. L., H. M. Pooley, and G. D. Shockman.
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15. 16.
17.
18.
19. 20. 21.
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1970. Site of initiation of cellular autolysis in Streptococcus faecalis as seen by electron microscopy. J. Bacteriol. 103:504-512. Huff, E., R. M. Cole, and T. S. Theodore. 1974. Lipoteichoic acid localization in mesosomal vesicles ofStaphylococcus aureus. J. Bacteriol. 120:273-281. Joseph, R., and G. D. Shockman. 1975. Cellular localization of lipoteichoic acid in Streptococcus faecalis. J. Bacteriol. 122:1375-1386. Leduc, E. H., and W. Bernhard. 1967. Recent modifications of the glycol methacrylate embedding procedure. J. Ultrastruct. Res. 19:196-199. Lewin, R. A. 1963. Rod-shaped particles in Saprospira. Nature (London) 198:103-104. McCandless, R. G., M. Cohen, G. M. Kalmanson, and L. B. Guze. 1968. Cores, microbial organelles possibly specific to group D streptococci. J. Bacteriol. 96:14001412. McCandless, R. G., T. J. Hensley, M. Cohen, G. M. Kalmanson, and L. B. Guze. 1971. A study of specificity of cores for group D streptococci. J. Gen. Microbiol. 68:357-365: Mollenhauer, H. H. 1964. Plastic embedding mixtures for use in electron microscopy. Stain Technol. 39:111114. Monneron, A., and W. Bernhard. 1966. Action de certaines enzymes sur des tissus inclus en epon. J. Microsc. (Paris) 5:697-714. Moor, H., and K. Muhlethaler. 1963. Fine structure in frozen-etched yeas'. cells. J. Cell Biol. 17:609-628. Pooley, H. M., and G. D. Shockman. 1970. Relationship between the location of autolysin, cell wall synthesis,
22.
23. 24.
25.
26.
27. 28. 29.
and the development of resistance to cellular autolysis in Streptococcus faecalis after inhibition of protein synthesis. J. Bacteriol. 103:457-466. Pope, L., D. P. Yolton, and L. J. Rode. 1967. Appendages ofClostridium bifermentans spores. J. Bacteriol. 94:1206-1215. Reynolds, E. S. 1963. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17:208-212. Ryter, A., E. Kellenberger, A. Birch-Andersen, and 0. Maaloe. 1958. Etude au microscope electronique de plasmas contenant de l'acide desoxyribonucleique. 1. Les nucleoides des bacteries en croissance active. Z. Naturforsch. Teil B 13:597-605. Shockman, G. D., and J. T. Martin. 1968. Autolytic enzyme system of Streptococcus faecalis. IV. Electron microscopic observations of autolysin and lysozyme action. J. Bacteriol. 96:1803-1810. Walker, P. D., and J. Short. 1968. The location of mucopolysaccharides on ultrathin sections of bacteria by the silver methanamine staining technique. J. Gen. Microbiol. 52:467-471. Weintraub, M. H., W. J. Ragetli, and B. Schroeder. 1971. The protein composition of nuclear crystals in leaf cells. Am. J. Bot. 58:182-190. Yamamoto, T. 1967. Presence of rhapidosomes in various species of bacteria and their morphological characteristics. J. Bacteriol. 94:1746-1756. Yolton, D. P., L. Pope, M. G. Williams, and L. J. Rode. 1968. Further electron microscope characterization of spore appendages of Clostridium bifermentans. J. Bacteriol. 95:231-238.
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Vol. 129, No. 1 Printed in U.S.A.
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Copyright X) 1977 American Society for Microbiology
Ultrastructural, Physiological, and Cytochemical Characterization of Cores in Group D Streptococcil SYLVIA E. COLEMAN2* AND ARNOLD S. BLEIWEIS Department of Microbiology and Cell Science, University ofFlorida, Gainesville, Florida 32611 Received for publication 3 August 1976
Cores are large, rod-shaped structures that have been found almost exclusively in group D streptococci, measure 0.1 to 0.16 ,um in diameter, and extend the width or length of cells. This study has shown that cores are produced in the cells at a reproducible point in early stationary growth after extensive mesosomal formation and after the pH has dropped below 6.5. When cells containing cores were introduced into a fresh medium with a pH above 6.5, the structures disappeared within 5 min. The structures were not found in young, logarithmically growing cells but formed in these cells upon autolysis or treatment with penicillin. Cores that were forming or disintegrating appeared to have a lamellar substructure. When chloramphenicol was added to the medium before the culture reached stationary phase, no cores were found in the cells. Cytochemical studies indicated that cores contain protein and are not composed of cell wall material or other polysaccharides that contain 1,2-glycol groups.
Cores are large, hollow, cylindrical structures that have been found almost exclusively in group D streptococci (16, 17) and were first observed by Abrams et al. (1) in 1964. These structures extend the width or length of a dividing cell and measure 0.1 to 0.16 ,um in diameter, with a tube wall that is between 0.01 and 0.03 ,um thick (3, 17). Cores normally form in stationary-phase cultures and have been observed after treatment with penicillin (R. G. McCandless, M. Cohen, G. M. Kalmanson, and L. B. Guze, J. Ultrastruct. Res. 21:162-163, 1967). Little is known of the chemical or physiological properties of these unusual structures. The work reported here shows that cores: (i) form at a reproducible point in early stationary growth phase after a period of extensive mesosomal formation; (ii) form upon autolysis or upon treatment with penicillin; (iii) are composed of protein and probably little or no carbohydrate; (iv) form in a growing culture only after the pH drops below 6.5; (v) do not form in the presence of chloramphenicol (CAP); and (vi) possess a lamellar substructure observable in cores that appear to be forming or disintegrating. The function of these structures has not been defined, but it is postulated that they may repre-
sent a labile repository for cellular protein, perhaps membranous in origin.
I Authorized for publication as paper no. 6174 in the Journal Series of the Florida Agricultural Experiment Station. 2 Present address: Research Service 151, Veterans Administration Hospital, Gainesville, FL 32602.
445
MATERIALS AND METHODS Cultures and media. Two strains of group D streptococci were used: Streptococcus faecalis ATCC 8043 and S. faecalis XL, a laboratory isolate. The bacteria were cultured in Todd-Hewitt broth (Difco), adjusted to a final pH of 7.2, and on agar plates of Todd-Hewitt broth with 2% agar added. Balanced cultural growth. Cultures were grown under conditions in which absorbance and dry weight were increasing at the same rate, and the lag phase was eliminated. Initial inocula of 0.1 ml of an overnight broth culture were spread on plates of Todd-Hewitt agar, which were incubated at 37°C overnight. Washings from a plate were inoculated into a flask containing 100 ml of broth prewarmed to 37°C and incubated to mid-logarithmic growth phase. From this culture, 30 ml was inoculated into a second prewarmed flask of 300 ml, and sequential inocula of 30 ml taken from mid-logarithmic phase were put into 300 ml of broth until balanced cultural growth was obtained. Dry weights were determined by filtering measured amounts of broth through a Nuclepore filter (General Electric Co.) with a pore size of 0.5 ,um and drying and weighing the filters. Turbidity was measured on a Gilford spectrophotometer at 660 nm. Cytochemical determination of polysaccharides. The presence of polysaccharides containing 1,2-glycol groups was detected in thin sections by the silver methenamine method (9, 26). The bacteria were fixed with glutaraldehyde and osmium tetroxide and embedded in an Epon-Araldite plastic mixture (18), and thin sections were placed on stainless-steel
446
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grids for staining. After oxidation with 1% periodic acid for 5 min at room temperature, the grids were washed and placed in the staining solution (100 ml of 3% hexamethylenetetramine [Eastman Organic Chemicals, Rochester, N.Y.] and 5 ml of 5% silver nitrate, both in double-distilled water). Enzyme digestion of fixed preparations. Bacteria were fixed in 2% glutaraldehyde in the cold and then embedded in water-soluble glycol-methacrylate (14). Thin sections were placed on stainless-steel grids and oxidized for 1 min with 3% hydrogen peroxide before exposure to the following enzymes (19,27) at 37°C: (i) Pronase (type VI, Sigma Chemical Co., St. Louis, Mo.), 0.2% in 0.2 M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride buffer (pH 7.4) for 10, 20, 30, and 45 min; and (ii) lysozyme (egg white, grade 1, Sigma Chemical Co.), 25,000 ,ug per ml in 0.01 M Tris-hydrochloride buffer (pH 8.3) for 30 min, 1, 2, and 6 h, followed by treatment with 2% sodium dodecyl sulfate for 10 min. Induction of cores by autolysis. Young cells from logarithmically growing cultures were harvested by centrifugation, washed three times with distilled water, and allowed to autolyze in 0.01 M potassium phosphate buffer (pH 6.5) at 37°C for periods of 15, 30, and 60 min (25). Cells in the process of autolysis were examined for core formation by electron microscopy. Induction of cores by penicillin. Cores were induced in cells from logarithmically growing cultures by the addition of 1,000 U of potassium penicillin G (Eli Lilly and Co., Indianapolis, Ind.) per ml for periods of 5, 10, 30, and 45 min, after which samples were fixed and embedded for observation with the electron microscope. Formation of spheroplasts. Spheroplasts of cells containing cores were prepared by the method of Coleman et al. (5, 6) by taking bacteria from stationary-phase culture and washing and incubating them for 30 min at 37°C with 250 jig of lysozyme per ml. Effect of pH on stability of cores. Cells and spheroplasts were incubated for 30 and 60 min at 37°C in the following buffers, with 0.5 M sucrose added for spheroplasts: 0.1 M HCl-KCl buffer (pH 2.0), 0.1 M citrate buffer (pH 4.0), 0.1 M potassium phosphate buffer (pH 7.0), 0.1 M Tris buffer (pH 7.2), 0.1 M potassium phosphate buffer (pH 8.0), and 0.1 M Tris buffer (pH 8.4). In experiments in which cells were grown in a buffered medium, Todd-Hewitt broth was prepared with 0.01 M potassium phosphate buffer (pH 7.2). Inhibition of core synthesis. The production of cores was inhibited by the addition of 50 Mug of CAP (Sigma Chemical Co.) per ml at 30 min or 1.5 h before formation of cores normally occurred in the culture. Electron microscopy. Various fixation techniques were used, including the method of Ryter et al. (24); various times of fixation were also used: 2% glutaraldehyde was utilized alone or followed by 1% osmium tetroxide, both in the cold and at room temperature, and sometimes preparations were postfixed with 2% uranyl acetate in buffer at room temperature. Prefixation with 0.01% osmium tetroxide, followed by fixation with glutaraldehyde
J. BACTERIOL.
and postfixation with osmium tetroxide and uranyl acetate, was also used with either sodium cacodylate or Veronal buffers. Embedding was in either an Epon-Araldite plastic mixture (18) or in Araldite (8). Thin sections were cut with a diamond knife (I. E. du Pont de Nemours & Co., Wilmington, Del.) on a Porter-Blum MT-2 ultramicrotome (Ivan Sorvall, Inc., Norwalk, Conn.), poststained with uranyl acetate and lead citrate (23), and observed with an Hitachi HUll-E electron microscope (Hitachi, Ltd., Tokyo, Japan) at 75 kV. Freeze-fracture. Fixed and unfixed bacteria were treated with 30% glycerol for 20 min, frozen in liquid Freon 22, and fractured at - 100°C on a Balzers BA 360 M freeze-etch apparatus (Balzers AG, Furstentum, Liechtenstein) using the method of Moor and Muhlethaler (20). The cells were sometimes etched for 2 or 4 min. The replicas were cleaned overnight in 40% aqueous chromium trioxide, washed several times in water, and picked up on uncoated grids.
RESULTS Structure of cores in ultrathin sections and in freeze-fractured cells. Good preservation of the core structures was obtained by fixation in glutaraldehyde alone or by various combinations of glutaraldehyde and osmium tetroxide. Figure la shows a cross section and Fig. lb a longitudinal section of cores in S. faecalis XL after fixation with glutaraldehyde in the cold followed by osmium tetroxide at room temperature. The appearance of the cores after freezefracture of either unfixed (Fig. lc) or fixed cells (Fig. ld) was similar to their appearance in thin section. Cores in S. faecalis ATCC 8043 differed from those in S. faecalis XL by being denser in cross section (Fig. 2a) and more fibrous in appearance (Fig. 2b). The structure of these cores in freeze-fractured cells (Fig. 2c and d) is again similar to their morphology in thin section. Formation of cores during balanced growth. S. faecalis XL was cultured under conditions in which the lag phase was eliminated and the absorbance and dry weight of the cells were increasing at the same rate. Under these conditions, cores appeared in the cells after 3.5h growth and formed reproducibly at this point in the growth cycle (Fig. 3). During h 1 of growth, few mesosomes were observed, but beginning at 2 h extensive mesosomal membrane was observed in the cells (Fig. 4). Numerous observations of cells in many thin sections indicated that mesosomes became noticeably smaller after the formation of cores at 3.5-h growth. Persistence of core structures and their relationship to viability of the cells. Cores and mesosomes could be observed in the cells throughout 7 days of growth in Todd-Hewitt
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FIG. 1. (a) Cross section of a core in S. faecalis XL after 20-h growth in Todd-Hewitt broth. Fixation in glutaraldehyde in the cold followed by osmium tetroxide at room temperature. Bar = 0.1 um. (b) Longitudinal section of a core in S. faecalis XL after 8-h growth in Todd-Hewitt broth and fixation in glutaraldehyde in the cold followed by osmium tetroxide at room temperature. Bar = 0.1 ,um. (c) Freeze-etched unfixed cell of S. faecalis XL after 36-h growth in Todd-Hewitt broth. Inset is a cross fracture of an unfixed core. Bars = 0.1 ,um. (d) Freeze-etched cell of S. faecalis XL fixed with glutaraldehyde after 14-h growth in Todd-Hewitt broth. Inset is a cross fracture of a glutaraldehyde-fixed core. Bars = 0.1 ,um.
448
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COLEMAN AND BLEIWEIS
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FIG. 2. (a) Cross section of a core in S. faecalis ATCC 8043 fixed with glutaraldehyde and osmium tetroxide after 24-h growth in Todd-Hewitt broth. Bar = 0.2 gm. (b) Longitudinal section of a core in S. faecalis ATCC 8043 fixed with glutaraldehyde and osmium tetroxide after 24-h growth in Todd-Hewitt broth. Bar = 0.2 ,um. (c) Cross section ofa core in a freeze-etched cell ofS. faecalis ATCC 8043 after 24-h growth in Todd-Hewitt broth. Bar = 0.2 ,um. (d) Longitudinal fracture of a core in a freeze-etched cell of S. faecalis ATCC 8043 after 24-h growth in Todd-Hewitt broth. Bar = 0.1 Jim.
broth at 37°C. The pH of the culture decreased to about 6.2 at the time cores formed (Fig. 3) and remained this low or lower during the 7 days of growth. The formation of cores did not
affect viability as measured in colony-forming units (CFU) and a decrease in CFU was observed only after 7 days. From a count of 8.2 x 107 CFU per ml after 30 min, there was an
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increase to 1 x 109 CFU per ml after 1.5 h, which remained through 48 h. At this time the pH, which was initially 7.4, had decreased to 5.9. Approximately 90% of the cells in thin sections contained cores after 3.5-h growth. Effect of CAP on the formation of cores. Thirty minutes and 1.5 h before cores formed normally, 50 jig of CAP was added to the culture (Fig. 5). In neither case did cores form after 5-h incubation, although numerous cells 2.5 1.2
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449
contained cores in control cultures. Frequent accumulations of clear material were noted in the cells treated with CAP, but no cores were formed. When cells treated with CAP were inoculated into fresh medium or into filtered, spent broth from a stationary-phase culture and incubated for 24 h, cores subsequently were produced. Conditions of formation and disaggregation of cores. Since young, logarithmically growing cells do not contain cores, it was hypothesized that the structures might disappear soon after inoculation of older cells into fresh broth. Cores, which were numerous in cells taken from a stationary-phase culture, could no longer be observed 5 min after the cells were inoculated into fresh medium at pH 7.2. Cells for the inoculum in this experiment were collected at 37°C to avoid a temperature shift that might influence the disposition of the cores. Cores, however, were found to be stable at 2°C for 60 min, indicating that they do not disintegrate in the cold. Cores also remained stable after placing the cells into fresh or filtered, spent Todd-Hewitt both at pH 6.1 (Table 1). Young cells without cores, when introduced into spent medium or fresh medium at pH 6.1, did not form cores after 3.5 h even though the cells grew slowly. In control cultures, the pH dropped to 6.2
FIG. 4. Mesosomes could be observed in almost every cell in thin sections ofS. faecalis XL after 2-h growth in Todd-Hewitt broth. Fixation by the method of Ryter et al. (24). Bar = 1.0 ,utm.
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COLEMAN AND BLEIWEIS
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FIG. 5. Growth of cultures of S. faecalis XL in Todd-Hewitt broth at 37°C after addition of 50 pg of CAP per ml: (A) 1.5 h before the normal time for formation of cores; (B) 30 min before core formation occurs; and (C) a control culture. TABLE 1. Changes in cells of Streptococcus faecalis XL with and without cores after inoculation into fresh and spent Todd-Hewitt broth at 37°C Cells With cores
Broth Fresh medium, pH 7.2
With cores
Fresh medium, pH 6.1
With cores
Spent broth, pH 6.1
Without cores Without cores
Spent broth, pH 6.1 Fresh medium, pH 6.1
Cores Disappear within 5 min Remain stable in the cells Remain stable in the cells No cores formed within 3 h No cores formed within 3 h
decreased to 6.2, the stability of cores in cells of S. faecalis ATCC 8043 was tested in relation to pH. Cores in cells of S. faecalis ATCC 8043 were stable after incubation for 60 min at 37°C in 0.1 M HCl-KCl buffer (pH 2.0), even though much of the cytoplasm was lost (Fig. 7). Cells incubated for 30 min at 37°C in 0.1 M citrate buffer (pH 4.0) also contained stable cores. Cores were also clearly discernible in cells placed in distilled water at pH 6.0. However, when the pH of the culture medium was raised from 6.1 to 7.0 after cores had been produced, and the culture was incubated for 30 min at 37°C, cores were no longer visible. Cells with cores that were incubated for 30 min at 37°C in 0.1 M Tris buffer (pH 7.2) no longer contained cores, nor did cells after 30 min at 37°C in 0.1 M potassium phosphate buffer (pH 8.0), nor after raising the pH of the growth medium to 8.0 for 30 min at 37°C. When the core-containing cells were chilled to 2°C for 30 min after raising the pH of the medium to 8.0, the core structures also disappeared, indicating that they were disrupted at the nonoptimal pH whether the cells were at growth temperature or in the cold. Even after a brief washing of 5 min in 0.1 M Tris buffer (pH 8.2), the cores were barely discernible. The cells remained viable after these treatments. Formation of cores in autolyzing cells and in the presence of penicillin. Young cells from
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after 2 h and was at this level at 3.5 h when cores were formed initially. In a culture of S. faecalis ATCC 8043 buffered in Todd-Hewitt broth with 0.01 M potassium phosphate buffer (pH 7.4) and incubated at 37°C, the growth was slower than normal, but after 48 h there was still no production of cores at a final pH of 6.6. Two cultures of S. faecalis XL buffered with 0.01 M citrate buffer (pH 4.5) and 0.1 M Tris buffer (pH 7.9) (Fig. 6) did not produce cells that formed cores: the former had diminished growth, and the pH of the latter only decreased to 6.5, which was apparently not low enough for the formation of cores. Effect of pH on the stability of cores. Since cores did not appear in culture until the pH
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FIG. 6. Growth of cultures of S. faecalis XL in: (A) unbuffered Todd-Hewitt broth; (B) Todd-Hewitt broth buffered with 0.01 M citrate buffer, pH 4.5; and (C) Todd-Hewitt broth buffered with 0.01 M Tris buffer, pH 7.9. Cultures were incubated at 37°C.
CORES IN GROUP D STREPTOCOCCI
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logarithmically growing cultures never contained cores. However, when these cells were permitted to autolyze in 0.1 M potassium phosphate buffer (pH 6.5) at 37°C, cores were formed as autolysis proceeded. Autolysis began in the septal area (11) and, after 15 to 30 min, large accumulations of granular material formed in the septal area of cells of S. faecalis ATCC 8043 (Fig. 8a). The structure of the cores that formed beginning at 30-min autolysis can be seen in S. faecalis ATCC 8043 (Fig. 8b). After 60-min autolysis, the cells began to lyse. Cores also could be induced in young cells by adding 1,000 U of penicillin G per ml to cultures at mid-logarithmic growth and incubating them an additional 30 min. Structure of cores in spheroplasts. The treatment of core-containing cells of S. faecalis XL with 250 ,ug of lysozyme per ml, at 37°C in distilled water at pH 6.0 for the formation of spheroplasts, resulted in the apparent disintegration of the core structure after only 5-min treatment with lysozyme. The cores degraded into layers of dense material (Fig. 9a and b). The layered substructure in the cores of spheroplasts was also evident in freeze-fractured preparations of unfixed spheroplasts (Fig. 9c and d). Intermediate forms of cores. Occasional forms could be seen in thin section (Fig. 10a) and in freeze-fracture (Fig. 10b) of older cells that appeared to be intermediates of cores, sim-
ilar to those seen in penicillin-induced spheroplasts. Cytochemical and enzymatic studies of core structure. To determine the possible presence in the core structures of polysaccharides containing 1,2-glycol groups, the silver methenamine staining procedure was used. Although the cell walls stained heavily (Fig. Ila and b), the core structures did not contain polysaccharides of these specifications. Cores disappeared from thin sections of cells embedded in glycol-methacrylate after digestion with Pronase for 45 min (Fig. 12a). This suggests the presence of a protein component in the cores. Digestion of sections with 25,000 ,ug of lysozyme per ml (Fig. 12b) removed the cell wall but not the core structure. This suggests that substantial amounts of peptidoglycan are not present in the core. Digestion of cores was also attempted with 0.1 % ribonuclease, deoxyribonuclease, lipase, and 0.5% trypsin with inconsistent or negative results.
DISCUSSION The fact that the structure of the core was the same in fixed, embedded cells and in freezefractured, unfixed cells established a basis for further work on the properties, development, and stability of these unusual structures. The
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COLEMAN AND BLEIWEIS
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FIG. 8. (a) Section showing accumulations of material near septal area of S. faecalis ATCC 8043 after 30min autolysis in 0.01 M phosphate buffer, pH 6.5. Bar = 0.1 um. (b) Thin section ofa core forming in a cell of S. faecalis ATCC 8043 after autolysis for 30 min at 37°C in 0.01 Mphosphate buffer, pH 6.5. Bar = 0.1 ,um.
main chemical and physical characteristics of and Smith (7) postulated that the microtubular cores are their lability at higher pH and the structures that they observed in L-forms of S. apparent protein nature of the structures. Dis- faecalis var. zymogenes could be aberrant cernible subunits were not observed in the forms of the normal mesosome that were no cores, although most other tubular structures longer observed in bacterial protoplasts and Lin prokaryotic cells, such as gas vacuoles (2, 4), forms. It may be that mesosomal membrane in spore appendages (22, 29), and rhapidosomes (15, 28), have a regular arrangement of sub- group D streptococci has a structural specificity units. Layers of substructural material could be that causes it to disassemble and reassemble in observed both in cores forming in autolyzing a tubular configuration. The mesosomes in S. cells (Fig. 8b) and in cores that were apparently faecalis (10) occur in a baglike shape that could disintegrating in spheroplasts (Fig. 9a and b). be visualized as elongating into a tube (Fig. lOa The layers of protein-containing substructural and b). The problem involved in recognizing an components are apparently formed only when intermediate stage with certainty makes this the pH has decreased to 6.2, indicating that the difficult to investigate. protein is in an altered form at higher pH. It Higgins and Daneo-Moore (10) suggested also seems from the results of polysaccharide that the formation of mesosomal membrane in staining (Fig. lla and b) that cores do not con- S. faecalis ATCC 9790 depends upon the rate of tain cell wall components, as was suggested synthesis of deoxyribonucleic acid. During acearlier by Cohen et al. (3). tive, logarithmic growth, large amounts of mesSeveral workers have observed rodlike struc- osomal membrane are produced, as was obtures to which they attributed a membranous served in S. faecalis XL. When the cultures origin in streptococci and other bacteria. Kuhrt reached stationary growth phase, the rate of and Pate (Bacteriol. Proc., p. 165, 1971) ob- deoxyribonucleic acid synthesis slowed. It is served large amounts of mesosomal membrane shortly after this period that smaller mesosoin cells of Chondrococcus columnaris which, mal structures are seen (10). upon lysis, released large numbers of small Apparently, the specific protein(s) that goes microtubular structures which they concluded, into the core is present in the cells during logafrom a comparison of the chemical composi- rithmic growth but is arranged into a core only tions, were derived from the mesosomal mem- when growth is interrupted. This rearrangebrane. Studying group D streptococci, Corfield ment to form a core does not occur when protein
VOL. 129, 1977
CORES IN GROUP D STREPTOCOCCI
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FIG. 9. (a) Cross section of an apparently disintegrating core in a spheroplast of S. faecalis XL after exposure to 250 pg of lysozyme per ml for 30 min at 37°C in distilled water + 1% glucose at pH 6.0. Bar = 0.2 p.m. (b) Longitudinal section showing filamentous structure of an apparently disintegrating core in a spheroplast of S. faecalis XL after exposure to 250 pg of lysozyme per ml for 30 min at 37°C in distilled water + 1% glucose, pH 6.0. Bar = 0.2 pm. (c) Freeze-etched preparation showing a longitudinal fracture of a disintegrating core in a spheroplast ofS. faecalis XL after exposure to 250 pg of lysozyme per ml for 30 min at 37°C in the presence of 0.5 M sucrose. Bar = 0.2 ,um. (d) Cross fracture of a core in a freeze-etched spheroplast of S. faecalis XL after exposure to 250 pg of lysozyme per ml for 30 min in 0.5 M sucrose. Bar = 0.2 ,um.
synthesis is inhibited by CAP, indicating that some factor involved in the formation of cores must be affected by CAP. There are several possible explanations for this. First, perhaps
there is an enzyme(s) or structural protein that must be produced de novo as the core is formed. Second, mesosomal membrane is rarely seen in cells treated with CAP, and cores would not be
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COLEMAN AND BLEIWEIS
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9
FIG. 10. (a) Section ofa possible intermediate form of a core in S. faecalis ATCC 8043. Bar = 0.2 um. (b) Freeze-etched cell of S. faecalis XL showing a possible intermediate form of a core. Bar = 0.2 p.tm. _t
FIG. 11. (a) Silver methenamine stain of a section of S. faecalis XL showing heavy deposition of stain in the cell wall and none in the longitudinal core structure. Bar = 0.1 pm. (b) Cross section of a core unstained by the silver methenamine method with heavy staining of the cell wall polysaccharide. Bar = 0.2 pum.
formed if they were derived from mesosomal membrane whose synthesis was inhibited by CAP. The third possibility is derived from the work of Pooley and Shockman (21), who found
over an 80% decrease in autolytic activity of cells of S. faecalis ATCC 9790 within 10 min after treatment of cells with 100 ug of CAP per ml. If cellular autolysis is involved in the pro-
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FIG. 12. (a) Section of glutaraldehyde-ftxed S. faecalis XL showing digestion of the core structure after treatment of the section with 0.2% Pronase in 0.2 M Tris-hydrochloride buffer (pH 7.4) for 45 min at 37°C. Bar = 0.2 pum. (b) Section of glutaraldehyde-fixed S. faecalis XL showing digestion of the cell wall but no effect on the core structure after exposure to 25,000 pg of lysozyme per ml in 0.01 M Tris-hydrochloride buffer (pH 8.3) for 60 min at 37°C. Bar = 0.2 pum.
duction of cores, then CAP could be inhibiting the process by interfering with autolysis. Recent studies with S. faecalis have revealed that lipoteichoic acid (LTA) is secreted into the medium during formation of osmotically fragile bodies from exponential-phase cells (13). Evidence was obtained that the LTA was not associated with isolated mesosomal vesicles. In Staphylococcus aureus, however, Huff et al. (12) showed that mesosomal vesicles were the major location of LTA. In relation to core structures, it seems unlikely that the group D antigen, which has a-1, 2-linked glucose units (kojibiose) as the immunodeterminant group, would be present in any substantial quantity because of the failure of cores to stain by the silver methenamine method. However, this does not rule out some involvement (16) of LTA in the process of core formation. Immunocytochemical studies currently are in progress to verify the cellular localization of LTA in membranous structures of S. faecalis. ACKNOWLEDGMENTS We wish to thank H. C. Aldrich for his advice throughout this study and for the use of the facilities of the Ultrastructure Laboratory in the Department of Botany. We thank E. P. Previc for his critical reading of the manuscript and J. Duggan for help with the figures. This research was supported by grants from the Florida Agricultural Experiment Station (BC-01440) and a Public Health Service grant from the National Institute of Dental Research (DE-02901-08).
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