Vol. 122, No. 3 Printed in U.S.A.

JOURNAL OF BACTERIOLOGY, June 1975, p. 1375-1386 Copyright 0 1975 American Society for Microbiology

Cellular Localization of Lipoteichoic Acid in Streptococcus faecalis RONALD JOSEPH AND GERALD D. SHOCKMAN* Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140 Received for publication 3 March 1975

The release of lipoteichoic acid and mesosomal vesicles to the supematant buffer during the formation of spherical, osmotically fragile bodies was studied using Streptococcus faecalis ATCC 9790. Autolytic N-acetylmuramidase action was permitted to take place in exponential-phase cells incubated in a buffer which provides an exceptional degree of osmotic stabilization. Both lipoteichoic acid and mesosomal vesicles were relatively rapidly released to the supernatant buffer. Most of the cellular content of lipoteichoic acid (and mesosomal vesicles) was found in the supematant buffer at incubation times when the cells still retained over 75% of their cell wall. ["4C]- or [3H]glycerol was used as a label for both cellular lipoteichoic acids and lipid-glycerol. Glycerol in lipoteichoic acid was quantitated after phenol-water and chloroform-methanol treatments and identified by products of acid hydrolysis and its ability to be precipitated by (i) antibodies specific for the polyglycerol-phosphate backbone, (ii) antibodies to the streptococcal group D antigen, and (iii) concanavalin A. Evidence was obtained that lipoteichoic acid was not associated with isolated mesosomal vesicles. Centrifugation of supernates at 200,000 x g sedimented membranous (mesosomal) vesicles and nearly all of the lipid-glycerol present, whereas essentially all of the lipoteichoic acid remained in the supematant. The sedimented mesosomal vesicles differed from protoplast membrane in their higher lipid-phosphorus to protein ratio and in the absence of detectable levels of two enzymatic activities found in protoplast membranes, adenosine triphosphatase and polynucleotide phosphorylase. Both types of membranes were found to contain DD-carboxypeptidase and LD-transpeptidase activities at nearly the same specific activities. No evidence was obtained for the association of autolytic N-acetylmuramidase activity with either type of membrane preparation. In gram-positive bacteria, teichoic acids have been found either covalently linked to the cell wall peptidoglycan as wall teichoic acids and as so-called intracellular teichoic acids. All intracellular teichoic acids investigated so far are glycerol teichoic acids, whereas wall teichoic acid may contain glycerol or ribitol (2, 13, 41). In addition, when carefully examined, intracellular (glycerol) teichoic acids have been found to contain lipid, and therefore have been called lipoteichoic acids (13, 39, 41). Much of the available information on lipoteichoic acids has been summarized in recent reviews (13, 41). Several lines of evidence suggested that lipoteichoic acids are closely associated with the cell membrane (26, 28, and summarized in references 2 and 13). Recently, direct evidence that the lipoteichoic acid of Lactobacillus fermenti NCTC 6991 is associated with the outer surface of the protoplast membrane was ob-

tained via electron microscopy using the ferritin-labeled antibody technique (36). Steptococcus faecalis ATCC 9790 (S. faecium) possesses an intracellular glycerol teichoic acid which contains a 1,2-linked glucose units (i.e., kojibiose or kojitriose, the determinant group of the streptococcal group D antigen), linked to hydroxyl groups of its polyglycerolphosphate chains (38). A number of years ago experimental evidence was presented which indicated that most of this antigen was closely associated with the protoplast membrane of this organism (26, 28). Relatively small amounts of serologically reactive material were present both in washes of the membrane fraction and in the supematant buffer after hen egg-white lysozyme-catalyzed protoplast formation, in a system in which 0.5 M sucrose, 0.05 M sodium phosphate, pH 6.6, and 10 mM magnesium acetate were used. In contrast to our results,

1375

1376

JOSEPH AND SHOCKMAN

Hay et al. (8) and Smith and Shattock (29), using other strains of group D streptococci, concluded that the glycerol teichoic acid and the group D antigen, respectively, are located between the cell wall and the protoplast membrane. We have now reinvestigated the cellular localization of the lipoteichoic acid of S. faecalis utilizing a recently developed system (12) in which the action of the native, wall-bound autolytic N-acetylmuramidase (27) was used to prepare protoplasts (autoplasts) under conditions which provide an unusually high degree of osmotic protection. In exponential-phase cells of this species the action of the native autolysin results in the initiation of wall loss at the sides and tips of nascent cross walls followed by the sequential loss of peripheral wall (10). In the osmotically protective system used, loss of less than 25% of the wall, at and near nascent septa, was accompanied by the attainment of maximal osmotic fragility (12). Loss of cross wall was accompanied by extrusion of mesosomal vesicles to the supernatant fluid, along with much of the cellular content of lipoteichoic acid. MATERIALS AND METHODS Growth and autoplast formation. Exponentialphase cultures of S. faecalis ATCC 9790 was grown in a chemically defined medium as previously described (25). For some experiments, cultures were grown in the presence of ["IC ]glycerol (2.25 uCi/Mmol, 54 nmol/ ml), [2-3H]glycerol (15.3 uCi/mol, 54 nmol/ml), or ["4C glycerol plus [3Hlleucine (3 uCi/4mol, 225 nmol/ ml) for six or more generations to insure uniform labeling of cellular macromolecules (22). Cells were washed and suspended at a concentration of 2 to 4 mg/ml (dry weight) in autoplast buffer (AP buffer) which consisted of 0.04 M ammonium acetate, pH 7.0, 0.5 M sucrose (enzyme grade, Schwartz-Mann) and 1 mM magnesium acetate, and incubated at 37 C for various time intervals. In some experiments trypsin (2 ug/mg cellular [dry weight]; General Biochemicals) was added to the incubation mixture. Trypsin addition was followed by the addition of soybean trypsin inhibitor (2 Mgblsg of trypsin) 10 to 15 min later. Measurement of release of [3Hjleucine and ["4C]glycerol during autoplast formation. Washed cells which had been grown in the presence of [4C ]glycerol and [3H ]leucine were suspended in AP buffer and incubated at 37 C. At the beginning of the incubation and at selected time intervals thereafter, multiple 1.0-ml samples were withdrawn and centrifuged at 12,000 x g for 10 min. The samples were treated as follows: the samples for total radioactively labeled compounds in the pellets and supernatant fractions received no further treatment. For trichloroacetic acid-precipitable material, pellets and supernatant fluid were treated separately wvith 5 ml of icecold 10% trichloroacetic acid for 20 min, and filtered through glass-fiber filter (Reeve Angel 984-H). The material retained by the filters was washed five times with cold 10% trichloroacetic acid. For lipids, samples

J. BACTERIOL.

were extracted by the chloroform-methanol method of Toennies et al. (31). The lipoteichoic acid fraction was quantitatively recovered after the phenol extraction procedure of Wicken and Knox (39). Without further fractionation this procedure will not distinguish between lipoteichoic acid and its deacylated form (41). Therefore we have used the term "lipoteichoic acid" to include both of these forms of glycerol teichoic acid. Radioactivity in all fractions was determined as previously described (12, 22). All samples on glass fiber filters were dissolved by treatment with 0.5 ml of 90% NCS (Amersham/Searle). Electron microscopy was performed as previously described (12). Protein (15) and total phosphorus (14) were determined by the methods of Lowry et al. Rhamnose was determined by a micromodification (9) of the method of Dische and Shettles (5). Deoxyribonucleic acid and ribonucleic acid were determined as described by Toennies et al. (32). Adenosine triphosphatase was assayed by the method of Abrams et al. (1), and polynucleotide phosphorylase by the method of Munoz et al. (17). Hydrolysis and chromatography of teichoic acid. Samples of lipoteichoic acid fractions were hydrolyzed in 2 N HCl at 100 C for 3 h under nitrogen, evaporated to dryness in a stream of nitrogen gas at 37 C, dissolved in 50 gl of propan-1-ol and applied to Whatman no. 4 paper along with reference standards of compounds normally found in acid hydrolysates of glycerol teichoic acid. The paper was developed in propan-l-ol-ammonium hydroxide (specific gravity 0.88)-water, 6.3.1 (vol/vol) (7). After development and drying, papers containing radioactive samples were initially scanned for radioactive peaks and then were cut into 0.5-cm strips and counted in a Nuclear Chicago Mark I scintillation counter as described above. Concurrently run chtomatograms were sprayed with indicator sprays for periodate-oxidizable compounds (16), reducing compounds (35), ninhydrin-reactive compounds (0.25% ninhydrin in acetone) and phosphate (37). Reaction of glycerol lipoteichoic acid with antibody specific for polyglycerol phosphate. Samples of fractions which contained ["4C]- or [3H ]glycerollabeled lipoteichoic acid were mixed with 25 or 50 Ail of rabbit serum containing antibody specific for polyglycerol phosphate (24, 40; a gift from K. W. Knox) in the presence of 0.85% NaCl in a total of 250 ul. Optimal antigen-to-antibody ratios were not determined. The mixtures were incubated at 37 C for 1 h, and then at 4 C for 2 days. After incubation, 50 gl of goat anti-rabbit gamma globulin was added. The preparations were incubated at 4 C for 2 more h after which 1 ml of saturated ammonium sulfate was added and the incubation was continued overnight at 4 C. The tubes were centrifuged at 25,000 x g for 20 min and the pellet was washed twice with saturated ammonium sulfate. The pellets were dissolved in 0.5 ml of 90% NCS for 2 h and then placed in scintillation vials for counting. Precipitation of [14C]- or [3H]glycerol-labeled lipoteichoic acid with concanavalin A. Fractions containing glycerol-labeled lipoteichoic acid were mixed with concanavalin A (1 mg/ml; kindly provided by R. J. Doyle and D. Birdsell) and brought to 1 ml and a final concentration of 0.05 M tris(hydroxy-

VOL. 122, 1975

CELLULAR LOCALIZATION OF LIPOTEICHOIC ACID

methyl)aminomethane (Tris), pH 7.8, and incubated at 37 C for 1 h and then 24 h at room temperature. The preparations were centrifuged at 25,000 x g for 20 min. The pellets were dissolved in 0.5 ml of 90% NCS and counted as described above. Isolation of the mesosomal vesicles and protoplast membrane fractions. Mesosomal vesicles were isolated from the supernatant of partial autoplasts, prepared by incubating cells in AP buffer for 1.5 to 2 h. At this time of incubation, wall from the septal region of the cell was lost but 75% or more of the wall remained around the periphery of the cells (12). The partial autoplasts were sedimented at 12,000 x g for 20 min and mesosomal vesicles were sedimented from the supernatant buffer at 200,000 x g for 4 h. Phenolwater extractions for the determination of relative glycerol teichoic acid content were performed on crude, unwashed mesosomal vesicle preparations. Chemical composition and enzymatic activities (e.g., those shown in Table 6) were determined on preparations that were washed at least twice and, in some cases, fractionated on a 20 to 80% discontinuous sucrose gradient containing 0.04 M acetate, pH 6.7, and 1 mM Mg2+. Analyses of mesosomal vesicles on discontinuous sucrose gradients showed the presence of one major band near the interface between 30 and 40% sucrose. Incubation conditions used for the isolation of protoplast membrane preparations depended on the purpose of the experiment. For example, for determinations of the incorporation of [3H ]glycerol into glycerol teichoic acid-containing fractions (e.g., the experiment shown in Table 5), sedimented partial autoplasts, prepared as described above, were lysed by resuspension in 0.04 M acetate, pH 6.7, 1 mM Mg2+ containing ribonuclease (2 Ag/ml) and deoxyribonuclease (2 lAg/ml), and the insoluble (protoplast membrane plus residual wall), and soluble (cytoplasmic) fractions were separated by centrifugation (15,000 x g, 20 min). Thus, in these experiments the protoplast membrane fraction was grossly contaminated with cell wall material. For the determination of chemical and enzymatic constituents, the incubation in AP buffer was prolonged to 3 to 5 h. In a few experiments, incubations in AP buffer were preformed in the presence of trypsin. In these cases, trypsin (2 Ag/mg of cells) was added at the beginning of the incubation, soybean trypsin inhibitor (4 ,g/mg of cells) was added at 15 min, and the autoplasts were harvested at 90 min.

Both of these conditions yielded wall-free autoplasts (12) and differences in chemical or enzymatic constituents between protoplast membranes isolated from the two types of incubation conditions were not observed. Protoplast membrane fractions were isolated from sedimented and lysed autoplasts as described above. These membrane fractions were then washed at least twice in 0.04 M acetate, pH 6.7, containing 1 mM Mg2+. Fractionation of the protoplast membrane fraction on the 20 to 80% discontinuous sucrose gradient revealed the presence of one band near the interface between 50 and 60% sucrose.

RESULTS Release of [3H]leucine- and [t4CJglycerolcontaining compounds during autoplast for-

1377

mation. Exponential-phase cells were grown in the presence of [3H ]leucine and [ I 4C ]glycerol for 10 generations, washed, and then incubated in AP buffer at a concentration of 2 mg/ml, cellular dry weight. During the incubation nearly all of the [3H ]leucine in both the total or trichloroacetic acid-precipitable fractions was retained by the autoplasts (Table 1) and, even after prolonged incubation, only a small fraction (less than 11%) of the [3H]leucine was found in the supernatant fraction (Table 1, columns 5 and 6). Thus, during incubation in AP buffer, even for periods of up to 20 h, osmotic stability was maintained and only very little cellular protein was lost from the autoplasts. Essentially all (81 to 89%) of the 3H found in the supernatant fractions (as well as the 3H found in the pellets) was in macromolecules precipitable by cold trichloroacetic acid (Table 2). As expected, only 11,000 of 2,299,000 dpm of the [3H ]leucine incorporated, or 0.5% (Table 1, column 4), was found in the lipid fraction, and only a small fraction of the 3H in the lipid fraction was found in the supernatant fluid (Table 1, column 5). In contrast to the results with [3H ]leucine, a substantial fraction of the total ["4C]glycerollabeled cellular material was rapidly released into the supernatant fluid (Table 1). Apparently, suspension of the cells in AP buffer was sufficient to release 17.4% of the total ["4C ]glycerol (Table 1, column 5). Further incubation resulted in a relatively rapid increase in the fraction of the total 14C found in the supernatant fluid (Table 1, column 5) with maximal release of 42% of total cellular [14C]glycerol approached after 2 h. Whereas, in all samples, essentially all of the 14C in the autoplast pellets was precipitable by cold trichloroacetic acid (Table 1, column 2), only 14 to 19% of the 14C in the supernatants were precipitated by cold trichloroacetic acid (Table 2). Since, after release to the supernatant fluid, most of the [4C ]glycerol-labeled material was not precipitated by cold trichloroacetic acid, the sum (pellet plus supernatant fluid) of trichloroacetic acid-precipitable material (Table 1, column 4) decreased with time of incubation. Furthermore, whereas 65 to 78% of the [14C ]glycerol in the pellets was in the lipid fraction (Table 1, column 2), only 1 to 4% of the [14C ]glycerol-labeled material found in the supematant fractions was in the lipid fraction (Table 2). Thus, most of the ["4C]glycerol-containing substance(s) released to the supernatant during the incubation period appeared to be neither lipid nor precipitable by cold trichloroacetic acid. The difference between the total [14C ]glycerol and trichloroacetic acid-precipitable [4C ]glycerol found in the supernatant fluids

1378

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JOSEPH AND SHOCKMAN

TABLE 1. Release of [3H]leucine and

[I4C]glycerol labeled compounds to the supernatant fraction during autoplast formation

dpm (x 10-) in Fractions

Time (h) Pellet

[3H ]Ieucine Total

0

1

2 3 5

TriChlOrOaCetiC aCid

0

1 2

precipitable

3 5 20 Lipid

2 3 5

0

1

2 3 5

Trichloroacetic acid

0

1

precipitable

2 3 5 20 Lipid

0

1

2 3 5

Percentage of average of the Percentage of average of the c i, average deviation.

a

"

sum sum

76.1 94.3 139.5

2006 2506 2533 2133 2353 2245

1

["C ]glycerol Total

85.6 113.3 164.4 181.2 209.0

2085 2196 2221 2111 2129

0

Supernatant fluid

152.8 170 238

12.4 11.3 11.2 9.6 9.2

41.3 31.6 31.8 28.9 28.5

0.07 0.14 0.3 0.4 0.2

8.6 16.7 18.6 19.8 20.7

% In supernatant

Sum (pellet plus supernatant fluid)

2171 2309 2386 2292 2338 Avg. 2299 2082 2600 2673 2286 2523 2483 Avg. 2441 12.5 11.4 11.5 10.0 9.4 Avg. 11.0

Of each fractiona

Of total"

3.7 4.9 7.2 7.9 9.1 +

2%C 3.1 3.9 5.7 6.3 7.0 9.8

+

49.9 48.3 50.4 48.7 49.2 Avg. 49.3

6.1 6.6 7.4 10.4

7%C 0.7 1.3 1.6 3.6 2.0

+

3.3 4.1

0.003 0.006 0.013 0.017 0.008

9%C 17.4 33.9 37.7 40.2 42.0

+

1.4%c

41.3 36.5 35.4 31.0 32.9 30.0

1.53 2.29 3.58 3.45 3.02 3.01

42.83 38.79 38.99 34.45 35.92 33.01

10 8.4 9.1

27.1 22.5 20.7 21.5 22.2

0.14 0.3 0.48 0.76 0.23

27.24 22.8 21.18 22.26 22.43

0.5 1.3 2.3 3.4 1.0

3.6 5.9 9.2

3.1 4.6 7.3 7.0 6.1 6.1 0.3 0.6 1.0 1.5

0.5

for that fraction. of total.

(Table 1, column 5) must be considered to be trichloroacetic acid-soluble. Therefore, trichloroacetic acid-soluble material in the supernatant fractions after 2 to 3 h in AP buffer accounted for 31 to 33% of the total cellular content of ['H ]glycerol. Release of much of the glycerol-labeled material from cells required a period of incubation at

37 C and, presumably, autolytic damage to the septal portion of the wall (10, 12). Incubation at 0 C in AP buffer resulted in the release of 14 to 18% of the total glycerol label after 2 to 5 h (Table 3), whereas parallel incubations at 37 C resulted in release of 41 to 53% of the total [3H]glycerol. In general (Table 3), less [3H ]glycerol-labeled material was released

VOL. 122, 1975

when cells were incubated in water at 0 C (2 to 5% of the total 3H) than when cells were incubated in 0.5 M sucrose (14%) or in 0.04 M acetate containing 1 mM Mg2+ (7 to 9%). Furthermore, release of the [3H]glycerol label from cells at 0 C increased with incubation time when cells were placed in sucrose or in AP buffer but not in water. The osmotic shock of placing cells in solutions containing 0.5 M sucrose seemed to cause the release of some of the glycerol-containing substances. Further evidence suggesting that wall damage was needed for maximal release of substances containing the glycerol label from cells was obtained in experiments in which the latent form of the autolysin was proteinase-activated. Incubation of cells in AP buffer containing trypsin decreased the time at which maximal release of glycerol-containing substances was attained from 2 to 3 h to 10 to 15 min. Nature of glycerol-containing materials in the mesosomal vesicle fraction. Incubation of cells in AP buffer for periods for 2 to 3 h results in loss of nascent cross walls and expulsion of mesosomal vesicles to the supernatant fluid (12). Therefore, the 12,000 x g supernatant fractions, taken at 2 h in the experiment summarized in Table 1, were centrifuged at 200,000 x g for 20 h to sediment mesosomal vesicles. The 200,000 x g pellet contained mesosomal vesicles (Fig. 1A) and contained 29% of the total and 24% of the trichloroacetic acid-precipitable [8H ]leucine-labeled material present in the 12,000 g supernatant fluid (Table 4, column 7). About 0.6% of the sedimented [3H ]leucine was in the lipid fraction (Table 4, column 2). Only about 14% of the total ["4C ]glycerollabeled material in the 12,000 x g supernatant fluid was sedimented at 200,000 x g (Table 4, column 7). About 32% of the 14C in the 200,000 x g pellet was precipitated by trichloroacetic acid and about 21% was in the lipid fraction. Of the 86% of the total ["4C]glycerol which remained in the 200,000 x g supernatant fraction, only 6.7% was precipitated by trichloroacetic acid and about 0.6% was in the lipid fraction (Table 4, column 4). Thus, a relatively small amount of [4C ]glycerol-labeled substance was sedimented with the mesosomal vesicle fraction, some of which was in the lipid fraction, whereas the much larger portion of material containing [4C ]glycerol was not sedimented at 200,000 x g and was neither precipitated by trichloroacetic acid nor present in the lipid fraction. A molecular species which contains glycerol but which is not soluble in chloroform and is trichloroacetic acid-soluble is lipoteichoic acid. Isolation of ["4C]glycerol-labeled lipoteichx

1379

CELLULAR LOCALIZATION OF LIPOTEICHOIC ACID

oic acid. Lipoteichoic acids have been prepared by phenol-water extraction procedures (34, 39). Cells grown in the presence of a high specific activity of [2-3H]glycerol (0.5 ,Ci and 5 Asg/ml) for 10 generations were washed and incubated in AP buffer for 2 h. The partial autoplasts were sedimented by centrifugation (12,000 x g, 20 min) and the supematant fluid was then centrifuged at 200,000 x g for 4 h. The 200,000 x g supematant fluid was extracted with an equal volume of 90% phenol at 4 C for 1 h, the aqueous phase was removed and the phenol phase was washed with an equal volume of water. The combined aqueous layers were dialyzed, lyophilized, and extracted twice with chloroformmethanol (2:1) at 4 C for 2 h. After centrifugation (12,000 x g, 20 min) the chloroformmethanol was removed and the resultant crude lipoteichoic acid was dissolved in water. In some experiments the protoplast membrane fraction was recovered from lysed autoplasts as described in Materials and Methods and all four cell fractions (protoplast membrane, cytoplasm, 200,000 x g sedimented mesosomal vesicles and the 200,000 x g supernatant fraction) were treated as described above for the isolation of lipoteichoic acid. In the experiment summarized in Table 5, 76 x 104 dpm or 25% of [3H ]glycerol-labeled material was in the 200,000 x g supematant fraction. About 29 x 104 dpm or 38% of the total [3H]glycerol in this fraction was recovered in the lipoteichoic acid fraction after the phenolwater and chloroform-methanol treatments. Whereas the other cell fractions, and the protoTABLE 2. Percentages of [3H]leucine- and [14C glycerol-labeled compounds in supernatant fluids which were precipitated by trichloroacetic

acid or extracted by chloroform-methanol (lipid fraction)a TrichloroTime of incubation (h)

acetic acid-

Lipid

precipitable fraction

fraction (%)

88.9 83.2 84.9 84.3 81.3

0.1 0.1 0.2 0.2 0.1

17.7 13.7 19.2 17.4 14.6

1.6 1.8 2.6 3.8 1.1

(%)

[3H ]leucine 0 1 2 3 5

[14C ]glycerol 0

1 2 3 5 a

Data from experiment summarized in Table 1.

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JOSEPH AND SHOCKMAN

J. BAcTmioL.

TAL 3. Release of [3HJglycerol-Iabeled substances to the supernatant fraction during incubation of cells under various conditions Experiments

Temp (C)

Time

(min)

Total (dpm x 10-')

Supernatant fraction (dpm x 10-2)

Experiment 1 Cells in AP buffer (0.5 M sucrose, 0.04 M acetate, 1 mM Mg2+)

% of total

37

0 30 60 120

69

10.3 31.9 34.3 36.7

15.8 46.2 49.7 52.8

Cells in AP buffer

0

0 180

113

13.4 20.5

11.9 18.1

Cells in 0.04 M acetate plus 1 mM Mg2+

0

0 180

131

7.6 9.6

5.8 7.3

Cells in 0.5 M sucrose

0

130

Cells in water

0

0 180 0 180

11.6 18.2 4.6 3.1

8.9 14.0 3.5 2.3

Experiment 2 Cells in AP buffer

37

0 180

44.5

11.4 18.3

25.6 41.1

Cells in AP buffer

0

0 180

45.8

5.2 8.2

11.3 17.9

Cells in 0.04 M acetate plus 1 mM Mg2+

0

0 180

46.2

4.3 4.2

9.3 9.0

Cells in water

0

0 180

64.0

2.2 3.0

3.4 4.7

Experiment 3 Cells in AP buffer

37

0 30

57710

Cells in AP buffer

0

0

57710

30 300

plast membrane fraction in particular, contained substantial amounts of the glycerol label, only very small amounts of the [3H ]glycerol was recovered as lipoteichoic acid. Thus, 94% of the [3H ]glycerol found in lipoteichoic acid fractions from all cell fractions was recovered from the 200,000 x g supernatant fraction (Table 5, column 8). Only about 1% of the material recovered as lipoteichoic acid was found in the mesosomal vesicle fraction (Table 5, column 6). The amount of [3H Jglycerol recovered as lipoteichoic acid from the 200,000 x g supernatant fluid was about 9% of the total ['H ]glycerol incorporated by the cells. In other similar experiments, from 9 to 16% of the total incorporated [3H ]glycerol was recovered as lipoteichoic acid in 200,000 x g supematant fractions. Furthermore, when the supernatant fluid from cells

132

1859 15226

3.2 26.4

1859

3.2

3277 7964

5.7 14.0

merely placed in AP buffer at 0 C was subjected to this procedure, a substantial fraction of labeled glycerol was recovered as lipoteichoic acid, and the amounts recovered increased with time. For example, the [3H ]glycerol released to the supematant fluid was analyzed for content of lipoteichoic acid (Table 3). Lipoteichoic acid accounted for 23, 29, and 23% of the ['H]glycerol in supernatants of the samples incubated for 0, 30, and 300 min at 0 C, respectively, compared with 29% of the ['H]glycerol in the sample incubated for 30 min at 37 C. Thus, lipoteichoic acid was released in parallel with the release of total glycerol. Identification of products of acid hydrolysis of lipoteichoic acid fractions by paper chromatography. Samples of fractions presumed to contain [3H]glycerol-labeled lipo-

VOL. 122, 1975

CELLULAR LOCALIZATION OF LIPOTEICHOIC ACID

1381

FIG. 1. (A) Crude mesosome preparation after autoplasting and centrifugation of the 12,000 x g supernatant fluid at 200,000 x g for 2 h. Bar equals 100 nm. (B) Purified mesosomal membrane after sucrose density gradient centrifugation recovered from the 30 to 40% sucrose interface. The membranes consist of saccules with relatively smooth surfaces and protruding tubules. Bar equals 100 nm.

JOSEPH AND SHOCKMAN

1382

J. BACTERIOL.

TABLE 4. Presence of [3HJleucine- and [4C ]glycerol-labeled compounds in the 200,000 x g pellet and supernatant fractions of the supernatant of cells incubated in AP buffer for 2 h % of sum in pellet

Sum

Supernatant

Pellet

Fraction

[3H Jleucine Total Trichloroacetic acid precipitable

276 176

Lipid

683 658

64 0.6

1.6

[14C ]glycerol Total Trichloroacetic acidprecipitable Lipid

19 6

0.7

%oftotal

6.7

139 14

0.6

5

29 24

77

3.4

0.3

120 8

21

dpm x 10-2

959 734

97

1.8

32

4.3

%of total

dpm x 10-2

%oftotal

dpm x 10-2

47

0.4

14 43

10

86

3.4

TABLE 5. [3Hlglycerol in lipoteichoic acid in various cell fractions

membranes dpm X

Total After aqueous-phenol treat-

10-4

% of suma

dpm X

10-4

152 16.8

50 25.4

53 2.6

1.6

5.2

_C

200,000 x g

Mesosomal vesicles

Cytoplasm

Protoplast

Fraction

Sum

12,000 x g supematant

Autoplasts

% of Suma

17 3.9

dpm X

10-4

% of suma

supernate

dpm

% of

1o-

suma

X

25 4.7

8 7.1

76 42

25 63.5

0.3

1.0

29

94

dpm

10-

%

305 66.1

(100) 22

X

ment

After chloroform-methanol extraction (lipoteichoic

30.9

10

acid) aPercent of sum of dpm after each treatment. h Percent of sum of dpm in total after each treatment. c None detected.

teichoic acid were hydrolyzed in HCl and chromatographed as described under Materials and Methods. Three 3H-containing areas with Rt values of 0.18, 0.43 and 0.72 were observed. These Rf values corresponded to those reported for glycerol diphosphate, glycerol monophosphate and glycerol, respectively, in this solvent system (38). Phosphate was detected in the two slower moving spots. Concurrently run standards of glycerol monophosphate (detected by the presence of phosphate) and [4C ]glycerol (detected by radioactivity) yielded the reported Rt values. Alkaline silver nitrate detected the presence of only one reducing compound with the same Rt as that of an accompanying glucose standard. Ninhydrin revealed the presence of three compounds, with the largest, most intense spot at an Rf of 0.63, the same as that of an alanine standard. The two minor ninhydrinreactive compounds were not identified. Reaction of lipoteichoic acid-containing fractions with antibody specific for polyglycerol phosphate and with concanavalin A. Ring precipitation tests showed a visible precip-

itation reaction between lipoteichoic acid-containing fractions and antibody specific for the polyglycerol phosphate backbone. Attempts were made to quantitate this reaction by reacting 200 ul of the phenol-water and chloroformmethanol-extracted fraction from the 200,000 x g supernatant fraction in the experiment summarized in Table 5, with antibody to polyglycerol phosphate and with concanavalin A, as described under Materials and Methods. With the antibody, 3,570 dpm was found in the precipitate compared with 420 dpm in the control to which antipolyglycerol phosphate antibody was not added. The net amount precipitated was 12% of the 3H added. With concanavalin A, which reacts with the a 1,2 glucoside side chains of the lipoteichoic acid of S. faecalis, 5,260 dpm, or about 20% of the 'H added, was precipitated. The fractions of radioactivity precipitated must be considered to be indications of the minimal amounts present. Optimal ratios of the reactants were not determined. Furthermore, in the case of the antigenantibody reaction, it seems possible that the a

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CELLULAR LOCALIZATION OF LIPOTEICHOIC ACID

1,2 glycoside substituents (kojibiose and kojitriose) on the polyglycerol phosphate side chains may interfere with complete precipitation of the antigen (3). The lipoteichoic acid also reacted with Lancefield group D antibody, which is specific for 1,2-linked glucosyl units (3, 38). Isolation and some properties of the membranous (mesosomal) vesicles present in the 200,000 x g pellet of supernatant fluids of partial autoplasts. As described above, the 200,000 x g pellets of autoplast supernatant fractions contained membranous vesicles (Fig. 1A), protein and lipid glycerol but only extremely small amounts of the total cellular lipoteichoic acid (Table 5). When applied to a 20 to 80% discontinuous sucrose gradient and centrifuged at 150,000 x g for 2 h, mesosomal vesicles banded at the interface of 30 to 40% sucrose (Fig. 1B), whereas protoplast membranes, obtained from the osmotic lysis of autoplasts, banded at the interface of 50 to 60% sucrose. The greater buoyancy of mesosomal vesicle fractions was reflected by their lower content of protein (about 38%) than protoplast membranes (about 57%). The difference in protein content was accompained by a higher content of lipid phosphorus for mesosomal membrane (about 2.5%) than for protoplast membrane (about 0.7%), suggesting that the mesosomal vesicles had a higher lipid-toprotein ratio than protoplast membrane. Both types of membrane contained less than 1% deoxyribonucleic acid and in the range of 2 to 4% ribonucleic acid. The ribonucleic acid content of mesosomal membrane was consistently higher than that of protoplast membrane. Both types of membrane preparations consistently contained less than 0.7% rhamnose, indicating little contamination with cell wall. Only a few enzymatic activities of the membrane preparations were examined (Table 6). Both types of membranes contained DD-cara

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boxypeptidase and LD-transpeptidase activities (4), and the ratios of these two activities to each other were the same for both types of membrane. The differences in specific activities of these two enzymes in the two types of membrane was not considered to be significant especially when the lower protein content of mesosomal membrane was taken into account. Significant levels of ATPase and polynucleotide phosphorylase activity were found in the protoplast membrane but not in the mesosomal membrane. Polynucleotide phosphorylase activity was also found in the cytoplasmic fraction at about one-third the specific activity of that found in the protoplast membrane. Since the cytoplasm accounts for a much larger fraction of the cell than does the protoplast membrane (33), a large fraction of this activity must be considered to be cytoplasmic. A similar situation was reported for the polynucleotide phosphorylase activity in Micrococcus lysodeikticus (17, 23). Both types of membrane contained N-acetylmuramidase activity (Table 6). At 1 mM Mg2+, the specific activity found in both types of membrane was about the same. When autoplasts were prepared in the presence of 40 mM Mg2+ instead of 1 mM Mg2+, the specific activity of N-acetylmuramidase in isolated mesosomal vesicles was 6- to 13-fold higher than in protoplast membranes. However, even in the presence of 40 mM Mg2+, the total number of units of N-acetylmuramidase activity in the mesosomal vesicles was only 4% of the total activity recovered from the cells. Although the specific activity in the protoplast membrane was lower, this type of membrane contained a somewhat larger fraction of total cellular Nacetylmuramidase. However, at 1 mM Mg2+, over 90% of the activity was found in the wall fraction or, after complete wall hydrolysis, in the supernatant buffer (Joseph and Shockman, manuscript in preparation).

TABLE 6. Enzymatic activities in protoplast and mesosomal vesicles Enzyme activity

DD-carboxypeptidase (nmol/h per mg)a LD-transpeptidase (nmol/h per mg)a DD-carboxypeptidase activity LD-transpeptidase activity ATPase (nmol/min per mg) Polynucleotide phosphorylase (nmol/min/mg) N-acetylmuramidase (0.001 optical density/h per mg)

Mg'+ (mM)

Protoplast membrane

Mesosomal vesicles

1.0 1.0

218-440 0.9-1.9

129-143 0.5-0.56

231-242

230-286

115-290 10 18-35 46-108

0-20

1.0 1.0 1.0 40

a Assays kindly performed by J. Coyette as described in (4). membrane substance.

1 38 602

Specific activities are expressed per milligram of

JOSEPH AND SHOCKMAN

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DISCUSSION

The results presented here contrast with earlier studies with the same organism grown in the same chemically defined medium (26, 28). These earlier experiments were interpreted to indicate a membrane association of the strep-* tococcal group D antigen, with the antigen rather easily removed by washing. At that time a distinction between protoplast membrane and mesosomal vesicles was not made. Our earlier results and interpretation differed from that of Hay et al. (8) and of Smith and Shattock (29) who investigated the cellular localization of glycerol teichoic acid and of the group D antigen, respectively, in other group D strains. These two groups of investigators proposed that the glycerol teichoic acid group D antigen is located between the protoplast membrane and the cell wall. In our initial studies (28), serological (precipitin) techniques, using antibody to the streptococcal group D antigen, were used to localize the antigen in fractions of mechanically disrupted cells and in washed membranes isolated after lysozyme induction of protoplasts in 0.5 M sucrose, 0.05 M sodium phosphate, pH 6.6 Mg2+ was not added and the fragility of the protoplasts in this buffer system precluded their removal from the supernatant fluid before they were osmotically disrupted. Antigen was found in membrane preparations (which could well have contained mesosomal vesicles as well as protoplast membrane). Washing the membrane preparations did remove a substantial fraction of the antigen. The studies reported later (26) used the same osmotically stabilizing system for the preparation of lysozyme-induced protoplasts but with the addition of 10 mM Mg2+. The increased osmotic stability obtained permitted the separation of protoplasts and supernatant fluid (called lysozyme hydrolysate) by centrifugation. In these experiments (26) most of the serological reactivity was again found in the protoplast membrane fraction from which some could be removed by washings. Some antigen was found in the lysozyme hydrolysate, a portion of which was sedimented at 120,000 x g and therefore presumably in a mesosomal vesicle fraction. The 0.04 M sodium acetate, pH 7.0, 0.5 M sucrose buffer system used for the experiments we report here provides an unusual degree of osmotic stability even in the absence of added Mg2+ (12). In addition, at incubation times of 2 to 3 h in AP buffer containing 1 mM Mg2+, when nearly maximal amounts of lipoteichoic acid were found in the supernatant fluid (Ta-

J. BACTERIOL.

bles 1-4), most of the peripheral cell wall remained present (12) and presumably added to the protection of the protoplast membrane. Release of lipoteichoic acid along with septally associated mesosomal vesicles well before complete hydrolysis of the wall led to the supposition that the mesosomal vesicles might contain lipoteichoic acid. This did not prove to be the case, since centrifugation at 200,000 x g for as long as 20 h sedimented 86% of the lipid glycerol (Table 4) and mesosomal vesicles, but essentially all of the lipoteichoic acid remained in the supernatant fraction. Thus, our results with S. faecalis differ from those of Huff et al. (11) with Staphylococcus aureus. Huff et al. found about 80% of the cellular lipoteichoic acid of S. aureus in the mesosomal vesicle fraction and only 6% each in the plasma membrane and periplasm fractions. At present, it is difficult to reconcile the two sets of observations which utilize different organisms and many other different conditions. We can only add that when 20 mM Mg2+ (the Mg2+ concentration used in the laboratory of Huff, and collaborators [11, 30] is added to the AP buffer used in these studies, a relatively small and significant, but highly variable, fraction of lipoteichoic acid (and N-acetylmuramidase activity) was found in both protoplast membrane and mesosomal vesicle fractions (Joseph and Shockman, unpublished observations). Lipoteichoic acids are known to have a high affinity for membranes (including erythrocyte membranes; 13, 41). Mesosomal membranes differ from protoplast membrane in composition and other properties. Therefore, a perhaps somewhat tenuous possible explanation of the results of Huff et al. could be based on a higher affinity of lipoteichoic acids to bind to mesosomal vesicles than to protoplast membrane, especially in the presence of Mg2+, once the system has been disrupted. The very rapid release of substantial amounts of lipoteichoic acid when cells of S. faecalis were merely placed in 0.04 M sodium acetate, pH 7.0, plus 1.0 mM Mg2+ (Table 3) suggests that at least part of the cellular lipoteichoic acid is present very near the cell surface and is released through an intact, and apparently porous, cell wall. The observations of release of lipoteichoic acid from cells placed in 0.5 M sucrose and of the increased release in 0.04 M acetate, pH 7.0, 1 mM Mg2+ when 0.5 M sucrose was added (AP buffer), suggests that cellular plasmolysis may play a role. Alternately, during plasmolysis, extrusion of membranous material to the socalled periplasmic space, along with the concomitant collapse of the mesosomal sac (12) could be taken to be consistent with localization

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CELLULAR LOCALIZATION OF LIPOTEICHOIC ACID

of some lipoteichoic acid within the mesosomal sac and its expulsion to the periplasmic space with mesosomal vesicles. Such an interpretation would be consistent with the observations of Huff et al. (11). Also, the release of most of the cellular lipoteichoic acid at 2 to 3 h at 37 C in AP buffer, when wall loss is limited to a small amount of septally located wall, is consistent with the localization of lipoteichoic acid to the septal region and perhaps even within the mesosomal sac. However, the observed displacement by plasmolysis (12) of the intimate contact between wall and membrane normally seen in cell sections (10) could easily permit movement of peripherally or polarly located molecules towards the septal area. Although not vigorously disproven, the mesosomal localization of lipoteichoic acid as proposed by Huff et al. (11) seems unlikely for several reasons in addition to the results presented in this paper. First of all, are the observations of Van Driel et al. (36) on L. fermenti NCTC 6991 and Lactobacillus casei NCTC 6375. These investigators used electron microscopy and ferritin coupled to antipolyglycerol phosphate antibodies to demonstrate antigen-antibody reaction over much of the entire surface of cells of both species and, of the membrane surface of protoplasts of L. fermenti. In fact, heavy labeling with the ferritin conjugate was seen on the outside of the cell wall in L. fermenti. Recently, Dickson and Wicken (Proc. 8th Intern. Congr. Electron Microscopy, II, p. 114-115, 1974) used the ferritin labeling technique directly on thin sections of cells of Lactobacillus plantarum, L. casei, and Lactobacillus helveticus. Their electron micrographs clearly showed that labeling was not confined to mesosomes but occurred over a substantial portion of the protoplast membrane, in some cases reaching the surface of the wall. Secondly is the fact that several organisms such as L. fermenti and Streptococcus lactis are agglutinated by antipolyglycerol phosphate antibodies (13, 41). It would be rather difficult to visualize an antigenic determinant localized in either the mesosomal sac or mesosomal vesicles as being responsible for agglutination of cells. Similar to mesosomal vesicles prepared from several other species, mesosomal vesicles of S. faecalis appear to contain less protein and more lipid than protoplast membrane (6, 11, 20, 21, 23). The S. faecalis mesosomal vesicles are very similar in appearance in negatively stained preparations to the vesicles isolated from M. lysodeikticus (18, 19, 23). Similar to the case of M. lysodeikticus (6, 18), mesosomal vesicles from S. faecalis lack detectable levels of ATP-

1385

ase and polynucleotide phosphorylase, activities which are found in protoplast membranes (Table 6). Unlike the situation with M. lysodeikticus (19), an enrichment of the autolytic N-acetylmuramidase activity of S. faecalis is not found in either protoplast or mesosomal membrane. The presence of DD-carboxypeptidase and LD-transpeptidase activities (4) at similar specific activities and in similar ratios to one another in both protoplast membranes and mesosomal vesicles (Table 6), makes it unlikely that mesosomal vesicles are uniquely involved in peptidoglycan synthesis. Similar conclusions were reached by Reusch and Burger (20, 21) for Bacillus subtilis on the basis of the finding of higher specific activities of two other enzymatic activities involved in cell wall synthesis in protoplast membrane preparations than in mesosomal membranes. ACKNOWLEDGMENTS This investigation was supported by Research grant GB 20813 from the National Science Foundation and by Public Health Service research grant AI 05044 from the National Institute of Allergy and Infectious Disease. We thank J. Coyette for performing the DD-carboxypeptidase and LD-transpeptidase assays. We also thank A. J. Wicken for helpful discussions.

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