Vol. 126, No. 1 Printed in U.SA.
JOURNAL OF BACTERIOLOGY, Apr. 1976, p. 192-197 Copyright © 1976 American Society for Microbiology
Inhibition of Wall Autolysis in Streptococcus faecalis by Lipoteichoic Acid and Lipids R. F. CLEVELAND,* A. J. WICKEN, L. DANEO-MOORE, AND G. D. SHOCKMAN Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140
Received for publication 31 October 1975
Fully acylated lipoteichoic acid (LTA) isolated from Streptococcus faecalis ATCC 9790 (S. faecium) inhibited autolysis of walls from the same organism at concentrations (1.0 to 1.5 nmol of LTA per mg of wall) comparable to those found in intact cells. Partially deacylated LTA isolated from S. faecalis or chemically deacylated LTA failed to inhibit significantly in the same concentration range. Beef heart cardiolipin and commercially obtained dipalmitoyl phosphatidyl glycerol were also found to inhibit wall autolysis in S. faecalis. Chemical deacylation of beef heart cardiolipin also removed the inhibitory activity of this molecule. Lipid fractions isolated from S. faecalis that inhibited wall autolysis were: diphosphatidyl glycerol (cardiolipin), phosphatidyl glycerol, aminoacyl phosphatidyl glycerol, and a neutral lipid fraction. Glycolipids were not found to be effective inhibitors. The possible role of LTA and/or certain lipids as regulators of cellular autolytic activity is discussed. Bacterial autolysins have been implicated in the processes of surface growth and division of bacteria (7, 15, 19). Autolytic-defective mutants may exhibit altered surface growth and division characteristics (4). In addition, an active autolytic system appears required for the expression of antibiotic-induced cell lysis (16, 23). Since an outcome of autolytic activity is cell lysis, one or more regulatory mechanisms, operating at the cellular level, have been postulated (19). Streptococcus faecalis ATCC 9790 is a grampositive coccus that divides in a single plane. The organism contains a single autolytic enzyme activity, a ,3-1 -* 4-N-acetylmuramide glycan hydrolase (muramidase [21]). Hydrolytic activity has been associated with newly incorporated peptidoglycan (20) and appears to be regulated at the level of the intact cell (14, 17). Evidence suggesting that the capacity of cells to autolyze involves a substance (effector) operating, in a rapidly reversible fashion, at the level of activity of the autolytic enzyme has been presented (17). Lipoteichoic acids (LTAs), a class of compounds common to a wide variety of gram-positive bacterial species, consist of substituted polyglycerolphosphate chains joined covalently to glycolipid (31). In a previous preliminary study, purified preparations of LTAs isolated from two species of streptococci and two species of lactobacilli were found to inhibit the muramidase activities of both S. faecalis 9790 and Lactobacillus acidophilus (3). LTA also in-
hibited the N-acetylmuramyl-L-alanine amidase of Bacillus subtilis ATCC 6051. The amidase of Diplococcus pneumoniae R36A was not inhibited by LTAs obtained from the same species of streptococci and lactobacilli but was inhibited by a choline-containing Forssman antigen preparation from pneumococcus (8) that has been referred to as an LTA (6, 8). LTAs have been defined by Wicken and Knox as membrane-associated glycerol teichoic acids covalently linked to glycolipids (29). We have been unable to obtain a reaction of Forssman antigen (kindly provided by A. Tomasz) with antipolyglycerol phosphate antibody. Although pneumococcal F antigen appears to be similar in chemical composition to the wall teichoic acid of this species (2) and contains structurebound lipid, its classification as an LTA requires further study. Neither preparations of Forssman antigen of D. pneumoniae nor the membrane-associated succinylated mannan of Micrococcus luteus had an effect on the rates of wall lysis of S. faecalis. In the present paper we expand the previous studies to report that physiological concentrations of LTA isolated from S. faecalis 9790 are powerful inhibitors of the autolytic activity of S. faecalis. In addition, we have found that certain lipids inhibit autolysis in S. faecalis. MATERIALS AND METHODS Growth of cells. S. faecalis ATCC 9790 was grown in a chemically defined medium at 37 C (18). Cultures (10 to 20 liter) in mid-exponential phase (400 192
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jig [cellular dry weight] per ml) were poured over ice, and the cells were removed by centrifugation at 4 C in a continuous flow rotor. The cells were washed twice with cold doubly distilled water and lyophilized. Preparation of cell walls. Cells were disrupted by either of two methods. In the first, about 0.5 g of dried cells was suspended in 60 ml of 0.15 M sodium phosphate (pH 7.8) and disrupted by shaking with 60 ml of styrene divinylbenzene beads in a cell homogenizer (Rho Scientific Co.). Alternatively, cells suspended in 0.15 M sodium phosphate (pH 7.8) at a concentration of about 20 mg/ml were disrupted in a Ribi fractionator (Sorvall, Norwalk, Conn.). The wall-autolysin complex was isolated by differential centrifugation as described previously (21) and stored at -20 C in 0.15 M sodium phosphate (pH 7.8) after two washes in the same buffer. Prior to an experiment, a sample of the frozen suspension was thawed, and the walls were deposited by centrifugation at 18,000 x g for 20 min. Measurement of lysis of walls. The assay system for autolytic activity (21) consisted of 1.3 to 2.0 mg (dry weight) of wall-enzyme complex from exponential cells in a final volume of 3.0 ml of 0.01 M sodium phosphate (pH 6.7 to 6.8). Loss of turbidity of the wall suspensions was followed at 450 nm, and the rate of lysis was measured as a first-order reaction and is expressed per hour. Compounds to be tested were added as aqueous solutions or, in the case of hydrophobic substances, as solutions in an organic solvent. In the latter cases, the test was accompanied by a control consisting of an assay tube containing an equivalent volume of the same solvent. In these cases, inhibition of the autolytic rate was expressed relative to the appropriate control. Extraction and isolation of LTA from glycerollabeled cells. Two parallel cultures were grown for at least six doublings of turbidity (at a constant doubling time of 32 min) to a turbidity equivalent to about 300 ,ug (cellular dry weight)/ml, at which time chloramphenicol (10 ,ug/ml) was added, and the incubation was continued for 25 min. Chloramphenicol was added to prevent loss of cellular contents due to lysis during subsequent treatments. The smaller culture (200 ml) was grown in the presence of [3H]glycerol (10 ,uCi/ml, 0.02 umol/ml), whereas the larger culture (20 liters) was grown in the absence of this labeled compound. Cells from both cultures were separately harvested, and then the two cell pellets were combined. After four washes with distilled water at 0 C the pellet was suspended in 200 ml of distilled water, and the suspensions were exposed to a temperature of 100 C for 20 min. After centrifugation (4,000 x g, 20 min) the supernatant (hot-water extract) was lyophilized and suspended in 15 ml of distilled water. The pellet was suspended in 100 ml of distilled water and extracted with 100 ml of freshly distilled phenol in two 50-ml lots as described (28). The aqueous phase of the phenol extract was extracted with chloroform (to remove phenol), lyophilized, and dissolved in 2 ml of distilled water. Samples (2 ml) were fractionated on a Sepharose 6B column (2.5 by 33 cm, upward flow), followed by fractionation on a Bio-Gel P30 column (2.5 by 33 cm, downward flow)
LTA INHIBITION OF WALL AUTOLYSIS
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using 0.2 M ammonium acetate as eluant (24). Additional fractionation on the Bio-Gel P30 column was found to increase the resolution of peaks II and III (see Fig. 1). Samples (100 to 150 ul) from each of the 4-ml fractions collected were counted in Aquasol or Formula 947 (New England Nuclear). Fractions from each peak region were pooled, dialyzed against distilled water, lyophilized, and stored at -20 C. For individual experiments, the lyophilized material was dissolved in doubly distilled water at a concentration of 1.0 to 1.5 mg/ml. Suspensions kept frozen at -20 C were stable for at least 2 months. Lipid extraction. Parallel cultures of unlabeled cells (5 liters) and cells labeled with [3H]glycerol (50 ml, 10 ,uCi/ml) were grown to late exponential phase, harvested separately, washed once in 0.9% saline, and combined. The cells were then extracted with chloroform-methanol (2:1). Lipids were separated from nonlipid components by passage through a Sephadex G25 column (1.5 by 10 cm) as described previously (27), and the lipids were fractionated on a silicic acid column (25). The glycolipid and neutral lipid fractions were each dissolved in acetone. The crude phospholipid fraction was dissolved in chloroform-methanol (2:1). Phosphatidyl glycerol, diphosphatidyl glycerol (cardiolipin), and aminoacyl phosphatidyl glycerol fractions were obtained from the crude phospholipid fraction by chromatography on silicic acid paper (26) and were dissolved in either chloroform-methanol or ethanol. The concentration of lipid in each preparation was determined from the phosphorus content (1). Other procedures. Chemical deacylation of LTA was carried out at 37 C for 15 min in 0.2 M KOH in methanol followed by passage through a Dowex 50 (HI form) column (29). The concentration (nanomoles) of each LTA preparation was estimated from the phosphorus content (1) on the basis of an average 27.5 nmol of P/nmol of LTA (31). Dipalmitin (Sigma Chemical Co., St. Louis, Mo.), dipalmitoyl phosphatidyl glycerol (Serdary Research Labs, London, Ontario, Canada), monogalactosyl diglyceride (Analabs, Inc., North Haven, Conn.) and beef heart cardiolipin (Applied Science Labs, State College, Pa.) were dissolved in ethanol at concentrations of 5 to 7.5 mg/ml. Cardiolipin was deacylated in methanolic KOH as described above for LTA.
RESULTS Gel filtration of [3H]glycerol-labeled LTA of S. faecalis. The two successive extracts (hot water and hot aqueous phenol) of the cell residue each showed three peaks of 3H-containing material (Fig. 1). The first peak (peak I) eluted near the void volume of the column, as expected for fully acylated micellar LTA. The second peak (peak II) was included in the column and has a molecular size consistent with deacylated or partially deacylated (nonmicellar) LTA (9). The chemical nature of the 3H-labeled material in the third peak (peak III), which elutes close
194
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CLEVELAND ET AL.
500 200 100
x
45
0-
030
-
15
-
I
30
40
50 60 70 80 FRACTION NUMBER
90
FIG. 1. Elution profiles of[3H]glycerol on a Sepharose 6B and Bio-Gel P30 column system of hot-water and hot-aqueous-phenol extracts ofS. faecalis 9790. Counts are expressed as disintegrations per minute, and each profile reflects the total disintegrations per minute in the extract.
to the total volume of the double-column system, is not known. The hot-water extract contained a substantial level of peak II material (about 50% of total material), a lesser amount of peak III material (about 30%), and a relatively small amount of peak I material (about 20%). In contrast, the hot-aqueous-phenol extract contained very large amounts of peak I material (95% or more of total material) and only traces of peaks II and III. The total amount of 3H recovered in the aqueous phenol extract was about 10 times that in the hot-water extract. LTA extracted from S. faecalis 9790 with hot aqueous phenol has previously been shown to react with antibody specific for polyglycerol phosphate (10). Material from peaks I and II from the Sepharose 6B column has been shown to strongly react with streptococcal group D (kojibiose-specific antiserum [91). In addition, acid hydrolysis and paper chromatography of unfractionated LTA and samples of peak I and peak II from S. faecalis 9790 have shown the presence of [3H]glycerol-containing substances with the same mobility as glycerol, glycerol monophosphate, and glycerol diphosphate (9, 10).
Experiments performed using ['4C]acetate in addition to [3H]glycerol showed significant 14C in peak I but not in peak II (data not shown), which is consistent with the identification of peak I as acylated LTA and peak II as deacylated LTA. Effects of peaks I and II on wall lysis. The pooled fractions from peaks I and II (from hotaqueous-phenol extracts) were tested for their effect on lysis of cell walls. Material from peak I inhibited wall lysis at very low concentrations (Fig. 2). Only 36 nmol of phosphorus per mg of wall (equivalent to 1.3 nmol of LTA/mg of wall) was sufficient to give the maximum inhibition observed (70%). This ratio of LTA to wall is below the ratio of LTA to wall (3 to 5 nmol/mg) calculated to be present in cells of S. faecalis (3). Furthermore, we have found that in the same lysis test system peak I material is comparable in specific inhibitory activity to purified LTA from L. fermentum 6991, which has been chemically and serologically characterized (12). Neither peak II nor chemically deacylated peak I showed significant inhibition at concentrations up to four times the concentration of peak I that gave maximal inhibition. A much higher concentration of peak II gave some inhi-
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LTA INHIBITION OF WALL AUTOLYSIS
100 w
A):
0
o
Ao0
80 0
D 60
\* Peak I 0 Deacylated Peak I a
Peak 11
-J
0 c:-
_ t t* z 40 t 0 0 z
C 20 w
0.
0
I
l 20 3. 0l 1.0l.o 2.0 3.0
4.0 4.0
LTA (nmoles/mg wall) FIG 2. Effect of fully acylated (peak I), partially deacyllated (peak II), and chemically deacylated LTA from 'S. faecalis on autolysis of walls from S. faecalis. Peaks I and n were obtained from hot-aqueousphenoo1 extracts. Percent control rate is based on control rc:Pte constants of 0.2 to 0.6/h.
195
terial in peak III was partially (or slowly) dialyzable. Thus, it was not easily possible to remove salts from this fraction to quantitatively examine inhibitory activity in peak III in the absence of artifacts. Effects of lipids on wall lysis. A glycerol label in S. faecalis appears to be restricted primarily to lipids and LTA (13). Several observations, such as the loss of inhibitory activity upon deacylation of LTA and a suggestion of slight inhibitory activity in peak III (data not shown), led us to examine the effects of various lipids on lysis of the wall-enzyme complex. Both commercially available, highly purified lipid and lipid fractions extracted from S. faecalis were tested. Beef heart cardiolipin was a surprisingly effective inhibitor of wall lysis (Fig. 3). Substantial levels of inhibition, reaching a maximum of 80%, were attained at 10 to 20 nmol per mg of wall.
A comparison of the data shQwn in Fig. 2 and 3 shows that 55% inhibition was observed with either about 2 nmol of beef heart cardiolipin or about 1 nmol of peak I LTA per mg (dry weight) of wall. These observations suggest that LTA in ak I is abou pe autolysist as is andinhibitorDe-of wall is beefeffect heart cardiolipin.
bition (about 50% inhibition at 23.5 nmol/mg of wall). In contrast, high concentrations of chemically deacylated peak II tended to stimulate 100 wall lysis to a highly variable extent. For example, in one experiment 15.4 nmol of chemi- w cally deacylated LTA/mg of wall stimulated wall lysis by about 2.5-fold, whereas in another cexperiment (3.9 nmol/mg wall) lysis was stimu- o 80 lated by 16%. Hot-water extracts have been previously re- 0-J ported to inhibit autolysis in S. faecalis when added in amounts that were large as compared D 60 -J to the amounts used in the experiments re- 0 ported here (M. Sayare, L. Daneo-Moore, and G. D. Shockman, Abstr. Annu. Meet. Am. Z 40 Soc. Microbiol. 1972, G106, p. 48). Variable de- 0 grees of inhibition of wall lysis were obtained from unfractionated hot-water extracts in the wz experiments reported here. This was appar- c 20 ently due, at least in part, to the relatively low 0.w amounts of peak I material recovered in hotwater extracts. Peak III material from hot-water extracts was also tested for inhibitory activity. Assays of large amounts of partially dialyzed peak III material indicated the presence of a low level of inhibitory activity. However, fractionation of a hot-water extract of a culture grown in the presence of [3H]glycerol and ['4C]acetate showed that peak III contained more than one component (data not shown). Furthermore, ma-
0
0
20 10 30 40 PHOSPHOLIPID (nmoles/mg wall) FIG. 3. Effect ofbeef heart cardiolipin (acylated or chemically deacylated) and of various lipid fractions (from S. faecalis) on autolysis of walls of S. faecalis. PG, Phosphatidyl glycerol; DiPG, diphosphatidyl
glycerol (cardiolipin); aaPG, aminoacyl phosphatidyl glycerol.
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lated) LTA into the growth medium (9, 12), inhibition obtained is not much lower than that obtained with peak I LTA. As is the case for some other biological activities of LTA (31), deacylation removed inhibition of wall autolysis. Deacylated beef heart cardiolipin also was without inhibitory activity. The observation that inhibitory activity was lost after deacylation is of potential physiological significance, since it was shown recently that several microorganisms excrete deacylated or partially deacylated (as well as fully acylated) LTA into the growth medium (9, 12), whereas intact cells contain a predominance of acylated LTA (Fig. 1). The mechanism(s) of action of the inhibition reported here remains to be elucidated. It would be of interest to determine whether the inhibition is exerted on the wall substrate or on the enzyme. Also unclear at this time is the relationship between the inhibition obtained with LTA and that observed in the presence of certain lipids. However, the structural similarity between cardiolipin and LTA has been shown by experiments in which deacylated cardiolipin inhibits, to a variable extent, the reaction of LTA with antibodies specific to the polyglycerol phosphate backbone of various LTA preparations (30). In S. faecalis, LTA accounts for about 1 to 2% of the cellular dry weight (11), whereas phospholipids account for about 3.5% of the cellular dry weight (5, 22). Assuming an average of 25 to 30 phosphate residues per LTA and an average of 1 to 2 phosphate residues per lipid, respectively, it would appear that, per unit cellular dry weight, there is about two to three times more inhibitory potential from phospholipid TABLE 1. Effect of lipids from various sources on than from LTA. However, the high level oflipid wall lysis phosphorus in cells does not necessarily rule Con- out LTA as a regulatory molecule, especially o auto- since LTA can be excreted in both acylated and Lipid allwal trol wg/ *gm lytic rate deacylated forms (9). Removal of inhibitory activity by deacylation 92 20.9 1,2-Dipalmitin could provide a mechanism for the control of 42.1 86 autolytic activity, e.g., by action of a deacyl79.4 79 ase. It would not be unreasonable to assume 52 19.8 Dipalmitoyl phosphatidyl that deacylated LTA, which might no longer be glycerol 39.1 48 of use to the cell, would be excreted. In addi78.2 34 tion, the location of LTA on the outer surface of the protoplast membrane (10, 11), from which it 14.0 93 Monogalactosyl diglyceride is easily released and perhaps deacylated, 27.0 89 would facilitate interaction between this mole51.5 80 cule and a native autolysin functioning in adjacent areas of wall. Deacylation would prevent 132 94.9 Glycolipids (S. faecalis 9790) 36 lipophilic interactions between the lipid moiety Neutral lipids (S. faecalis 94.9 9790) of LTA and cellular membrane components 60 96.2 Phospholipids (S. faecalis that undoubtedly would be important in main9790) taining this configuration.
acylated beef heart cardiolipin at concentrations of 45 nmol/mg (dry weight) of wall failed to inhibit (or stimulate) wall autolysis. Dipalmitoyl phosphatidyl glycerol also inhibited wall lysis (Table 1), whereas 1,2-dipalmitin and a monogalactosyl diglyceride did not have significant inhibitory activity. For the last two compounds, the maximal levels of inhibition observed were significantly lower than those obtained with either phosphatidyl glycerol (Fig. 3) or cardiolipin (Table 1). It seems possible that the maximal achievable levels of inhibition were limited by the solubility of various lipids in the aqueous assay system used. A crude phospholipid fraction from S. faecalis inhibited wall autolysis (Table 1), as did the cardiolipin, phosphatidyl glycerol, and aminoacyl phosphatidyl glycerol fractions purified from the crude phospholipid fraction of S. faecalis (Fig. 3). The neutral lipid fraction of S. faecalis was also inhibitory (Table 1). In contrast, the crude glycolipid fraction obtained from S. faecalis was slightly stimulatory. DISCUSSION The present study demonstrates that LTA isolated from S. faecalis '9790 inhibited the muramidase activity of homologous organisms. The muramidase of S. faecalis was also inhibited by commercially obtainable lipids and by several of the lipids and phospholipids that are commonly found in bacteria. Interpretation of the quantitative data is limited by the solubility of various lipid molecules in the assay system. Nevertheless, it would appear that, at least for the phosphatidyl glycerol and cardiolipin fractions, the order of magnitude of the
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Studies of the kinetics of inhibition by both LTA and lipids as well as determination of the accumulation of each in various physiologically altered states are required. It is also entirely possible that more than one cellular constituent acts as a regulatory molecule of the muramidase of S. faecalis. In S. faecalis a single enzyme activity (21) is present to perform all of the physiological roles that have been assigned to bacterial autolysins (15, 19). Some of these roles may depend on autolysins located well within the wall, whereas others may require enzymatic action at the outer boundary of the wall. ACKNOWLEDGMENTS This work was supported by Public Health Service grant AI 05044 from the National Institute of Allergy and Infectious Diseases and grant GB 20813 from the National Science Foundation. A. J. W. was a senior foreign Fellow of the American Association of Dental Research. R. C. was supported by Public Health Service training grant CA 05280 from the National Cancer Institute. Excellent technical assistance from T. Greber is acknowledged. We thank B. Rosan for the use of his Ribi cell fractionator. LITERATURE CITED 1. Ames, B. N., and D. T. Dubin. 1960. The role of polyamines in the neutralization of bacteriophage deoxyribonucleic acid. J. Biol. Chem. 235:769. 2. Briles, E. B., and A. Tomasz. 1973. Pneumococcal Forssman antigen. A choline-containing lipoteichoic acid. J. Biol. Chem. 248:639.-6397. 3. Cleveland, R. F., J.-V. Holtje, A. J. Wicken, A. Tomasz, L. Daneo-Moore, and G. D. Shockman. 1975. Inhibition of bacterial wall lysins by lipoteichoic acids and related compounds. Biochem. Biophys. Res. Commun. 67:1128-1135. 4. Forsberg, C. W., P. B. Wyrick, J. B. Ward, and H. J. Rogers. 1973. Effect of phosphate limitation on the
5.
6. 7. 8. 9.
morphology and wall composition ofBacillus licheniformis and its phosphoglucomutase-deficient mutants. J. Bacteriol. 113:969-984. Ganfield, M.-C. W., and R. A. Pieringer. 1975. Phosphatidylkojibiosyl diglyceride. The covalently linked lipid constituent of the membrane lipoteichoic acid from Streptococcus faecalis (faecium) ATCC 9790. J. Biol. Chem. 250:702-709. Goebel, W. F., T. Shedlovsky, G. I. Lavin, and M. H. Adams. 1943. The heterophile antigen of pneumococcus. J. Biol. Chem. 148:1-15. Hartmann, R., J.-V. Holtje, and U. Schwarz. 1972. Targets of penicillin action in Escherichia coli. Nature (London) 235:426-429. Holtje, J.-V., and A. Tomasz. 1975. Lipoteichoic acid: a specific inhibitor of autolysin activity in pneumococcus. Proc. Natl. Acad. Sci. U.S.A. 72:1690-1694. Joseph, R., and G. D. Shockman. 1975. The synthesis and excretion of glycerol teichoic acid during growth of two streptococcal species. Infect. Immun. 12:333-
338. 10. Joseph, R., and G. D. Shockman. 1975. Cellular localization of lipoteichoic acid in Streptococcus faecalis. J. Bacteriol. 122:1375-1386. 11. Knox, K. W., and A. J. Wicken. 1973. Immunological properties of teichoic acids. Bacteriol. Rev. 37:215257.
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12. Markham, J. L., K. W. Knox, A. J. Wicken, and M. J. Hewett. 1975. Formation of extracellular lipoteichoic acid by oral streptococci and lactobacilli. Infect. Immun. 12:378-386. 13. Pieringer, R. A., and R. T. Ambron. 1973. A method for the specific labeling of the glycerol in glyceride-containing lipids of Streptococcus faecalis ATCC 9790. J. Lipid Res. 14:370-372. 14. Pooley, H. M., and G. D. Shockman. 1970. Relationship between the location of autolysin, cell wall synthesis, and the development of resistance to cellular autolysis in Streptococcus faecalis after inhibition of protein synthesis. J. Bacteriol. 103:457-466. 15. Rogers, H. J. 1970. Bacterial growth and the cell envelope. Bacteriol. Rev. 34:194-214. 16. Rogers, H. J., and C. W. Forsberg. 1971. Role of autolysins in the killing of bacteria by some bacterial antibiotics. J. Bacteriol. 108:1235-1243. 17. Sayare, M., L. Daneo-Moore, and G. D. Shockman. 1972. Influence of macromolecular biosynthesis on cellular autolysis in Streptococcus faecalis. J. Bacteriol. 112:337-344. 18. Shockman, G. D. 1963. Amino acids, p. 567-673. In F. Kavanagh (ed.), Analytical microbiology. Academic Press Inc., New York. 19. Shockman, G. D., L. Daneo-Moore, and M. L. Higgins. 1974. Problems of cell wall and membrane growth, enlargement and division. Ann. N.Y. Acad. Sci. 235:161-197. 20. Shockman, G. D., H. M. Pooley, and J. S. Thompson. 1967. Autolytic enzyme system of Streptococcus faecalis. III. Localization of the autolysin at the sites of cell wall synthesis. J. Bacteriol. 94:1525-1530. 21. Shockman, G. D., J. S. Thompson, and M. J. Conover. 1967. The autolytic enzyme system of Streptococcus faecalis. II. Partial characterization of the autolysin and its substrate. Biochemistry 6:1054-1065. 22. Toennies, G., G. D. Shockman, and J. J. Kolb. 1963. Differential effects of amino acid deficiencies on bacterial cytochemistry. Biochemistry 2:294-296. 23. Tomasz, A., A. Albino, and E. Zaneti. 1970. Multiple antibiotic resistance in a bacterium with suppressed autolytic system. Nature (London) 227:138-140. 24. Toon, P., P. E. Brown, and J. Baddiley. 1972. The lipid-teichoic acid complex in the cytoplasmic membrane of Streptococcus faecalis N.C.I.B. 8191. Biochem. J. 127:399-409. 25. Vorbeck, M. L., and G. V. Marinetti. 1965. Intracellular distribution and characterization of the lipids of Streptococcus faecalis (ATCC 9790). Biochemistry 4:296-305. 26. Vorbeck, M. L., and G. V. Marinetti. 1965. Separation of glycosyl diglycerides from phosphatides using silicic acid column chromatography. J. Lipid Res. 6:36. 27. Wells, M. A., and J. C. Dittmer. 1963. The use of Sephadex for the removal of nonlipid contaminants from lipid extracts. Biochemistry 2:1259-1263. 28. Wicken, A. J., J. W. Gibbens, and K. W. Knox. 1973. Comparative studies on the isolation of membrane lipoteichoic acid from Lactobacillus fermenti. J. Bacteriol. 113:365-372. 29. Wicken, A. J., K. W. Knox. 1970. Studies on the group F antigen of lactobacilli: isolation of a teichoic acidlipid complex from Lactobacillus fermenti NCTC 6991. J. Gen. Microbiol. 60:293-301. 30. Wicken, A. J., and K. W. Knox. 1971. A serological comparison of the membrane teichoic acids from lactobacilli of different serological groups. J. Gen. Microbiol. 67:251-254. 31. Wicken, A. J., and K. W. Knox. 1975. Lipoteichoic acids: a new class of bacterial antigen. Science 187:1161-1167.
JOURNAL OF BACTERIOLOGY, Apr. 1976, p. 192-197 Copyright © 1976 American Society for Microbiology
Inhibition of Wall Autolysis in Streptococcus faecalis by Lipoteichoic Acid and Lipids R. F. CLEVELAND,* A. J. WICKEN, L. DANEO-MOORE, AND G. D. SHOCKMAN Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140
Received for publication 31 October 1975
Fully acylated lipoteichoic acid (LTA) isolated from Streptococcus faecalis ATCC 9790 (S. faecium) inhibited autolysis of walls from the same organism at concentrations (1.0 to 1.5 nmol of LTA per mg of wall) comparable to those found in intact cells. Partially deacylated LTA isolated from S. faecalis or chemically deacylated LTA failed to inhibit significantly in the same concentration range. Beef heart cardiolipin and commercially obtained dipalmitoyl phosphatidyl glycerol were also found to inhibit wall autolysis in S. faecalis. Chemical deacylation of beef heart cardiolipin also removed the inhibitory activity of this molecule. Lipid fractions isolated from S. faecalis that inhibited wall autolysis were: diphosphatidyl glycerol (cardiolipin), phosphatidyl glycerol, aminoacyl phosphatidyl glycerol, and a neutral lipid fraction. Glycolipids were not found to be effective inhibitors. The possible role of LTA and/or certain lipids as regulators of cellular autolytic activity is discussed. Bacterial autolysins have been implicated in the processes of surface growth and division of bacteria (7, 15, 19). Autolytic-defective mutants may exhibit altered surface growth and division characteristics (4). In addition, an active autolytic system appears required for the expression of antibiotic-induced cell lysis (16, 23). Since an outcome of autolytic activity is cell lysis, one or more regulatory mechanisms, operating at the cellular level, have been postulated (19). Streptococcus faecalis ATCC 9790 is a grampositive coccus that divides in a single plane. The organism contains a single autolytic enzyme activity, a ,3-1 -* 4-N-acetylmuramide glycan hydrolase (muramidase [21]). Hydrolytic activity has been associated with newly incorporated peptidoglycan (20) and appears to be regulated at the level of the intact cell (14, 17). Evidence suggesting that the capacity of cells to autolyze involves a substance (effector) operating, in a rapidly reversible fashion, at the level of activity of the autolytic enzyme has been presented (17). Lipoteichoic acids (LTAs), a class of compounds common to a wide variety of gram-positive bacterial species, consist of substituted polyglycerolphosphate chains joined covalently to glycolipid (31). In a previous preliminary study, purified preparations of LTAs isolated from two species of streptococci and two species of lactobacilli were found to inhibit the muramidase activities of both S. faecalis 9790 and Lactobacillus acidophilus (3). LTA also in-
hibited the N-acetylmuramyl-L-alanine amidase of Bacillus subtilis ATCC 6051. The amidase of Diplococcus pneumoniae R36A was not inhibited by LTAs obtained from the same species of streptococci and lactobacilli but was inhibited by a choline-containing Forssman antigen preparation from pneumococcus (8) that has been referred to as an LTA (6, 8). LTAs have been defined by Wicken and Knox as membrane-associated glycerol teichoic acids covalently linked to glycolipids (29). We have been unable to obtain a reaction of Forssman antigen (kindly provided by A. Tomasz) with antipolyglycerol phosphate antibody. Although pneumococcal F antigen appears to be similar in chemical composition to the wall teichoic acid of this species (2) and contains structurebound lipid, its classification as an LTA requires further study. Neither preparations of Forssman antigen of D. pneumoniae nor the membrane-associated succinylated mannan of Micrococcus luteus had an effect on the rates of wall lysis of S. faecalis. In the present paper we expand the previous studies to report that physiological concentrations of LTA isolated from S. faecalis 9790 are powerful inhibitors of the autolytic activity of S. faecalis. In addition, we have found that certain lipids inhibit autolysis in S. faecalis. MATERIALS AND METHODS Growth of cells. S. faecalis ATCC 9790 was grown in a chemically defined medium at 37 C (18). Cultures (10 to 20 liter) in mid-exponential phase (400 192
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jig [cellular dry weight] per ml) were poured over ice, and the cells were removed by centrifugation at 4 C in a continuous flow rotor. The cells were washed twice with cold doubly distilled water and lyophilized. Preparation of cell walls. Cells were disrupted by either of two methods. In the first, about 0.5 g of dried cells was suspended in 60 ml of 0.15 M sodium phosphate (pH 7.8) and disrupted by shaking with 60 ml of styrene divinylbenzene beads in a cell homogenizer (Rho Scientific Co.). Alternatively, cells suspended in 0.15 M sodium phosphate (pH 7.8) at a concentration of about 20 mg/ml were disrupted in a Ribi fractionator (Sorvall, Norwalk, Conn.). The wall-autolysin complex was isolated by differential centrifugation as described previously (21) and stored at -20 C in 0.15 M sodium phosphate (pH 7.8) after two washes in the same buffer. Prior to an experiment, a sample of the frozen suspension was thawed, and the walls were deposited by centrifugation at 18,000 x g for 20 min. Measurement of lysis of walls. The assay system for autolytic activity (21) consisted of 1.3 to 2.0 mg (dry weight) of wall-enzyme complex from exponential cells in a final volume of 3.0 ml of 0.01 M sodium phosphate (pH 6.7 to 6.8). Loss of turbidity of the wall suspensions was followed at 450 nm, and the rate of lysis was measured as a first-order reaction and is expressed per hour. Compounds to be tested were added as aqueous solutions or, in the case of hydrophobic substances, as solutions in an organic solvent. In the latter cases, the test was accompanied by a control consisting of an assay tube containing an equivalent volume of the same solvent. In these cases, inhibition of the autolytic rate was expressed relative to the appropriate control. Extraction and isolation of LTA from glycerollabeled cells. Two parallel cultures were grown for at least six doublings of turbidity (at a constant doubling time of 32 min) to a turbidity equivalent to about 300 ,ug (cellular dry weight)/ml, at which time chloramphenicol (10 ,ug/ml) was added, and the incubation was continued for 25 min. Chloramphenicol was added to prevent loss of cellular contents due to lysis during subsequent treatments. The smaller culture (200 ml) was grown in the presence of [3H]glycerol (10 ,uCi/ml, 0.02 umol/ml), whereas the larger culture (20 liters) was grown in the absence of this labeled compound. Cells from both cultures were separately harvested, and then the two cell pellets were combined. After four washes with distilled water at 0 C the pellet was suspended in 200 ml of distilled water, and the suspensions were exposed to a temperature of 100 C for 20 min. After centrifugation (4,000 x g, 20 min) the supernatant (hot-water extract) was lyophilized and suspended in 15 ml of distilled water. The pellet was suspended in 100 ml of distilled water and extracted with 100 ml of freshly distilled phenol in two 50-ml lots as described (28). The aqueous phase of the phenol extract was extracted with chloroform (to remove phenol), lyophilized, and dissolved in 2 ml of distilled water. Samples (2 ml) were fractionated on a Sepharose 6B column (2.5 by 33 cm, upward flow), followed by fractionation on a Bio-Gel P30 column (2.5 by 33 cm, downward flow)
LTA INHIBITION OF WALL AUTOLYSIS
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using 0.2 M ammonium acetate as eluant (24). Additional fractionation on the Bio-Gel P30 column was found to increase the resolution of peaks II and III (see Fig. 1). Samples (100 to 150 ul) from each of the 4-ml fractions collected were counted in Aquasol or Formula 947 (New England Nuclear). Fractions from each peak region were pooled, dialyzed against distilled water, lyophilized, and stored at -20 C. For individual experiments, the lyophilized material was dissolved in doubly distilled water at a concentration of 1.0 to 1.5 mg/ml. Suspensions kept frozen at -20 C were stable for at least 2 months. Lipid extraction. Parallel cultures of unlabeled cells (5 liters) and cells labeled with [3H]glycerol (50 ml, 10 ,uCi/ml) were grown to late exponential phase, harvested separately, washed once in 0.9% saline, and combined. The cells were then extracted with chloroform-methanol (2:1). Lipids were separated from nonlipid components by passage through a Sephadex G25 column (1.5 by 10 cm) as described previously (27), and the lipids were fractionated on a silicic acid column (25). The glycolipid and neutral lipid fractions were each dissolved in acetone. The crude phospholipid fraction was dissolved in chloroform-methanol (2:1). Phosphatidyl glycerol, diphosphatidyl glycerol (cardiolipin), and aminoacyl phosphatidyl glycerol fractions were obtained from the crude phospholipid fraction by chromatography on silicic acid paper (26) and were dissolved in either chloroform-methanol or ethanol. The concentration of lipid in each preparation was determined from the phosphorus content (1). Other procedures. Chemical deacylation of LTA was carried out at 37 C for 15 min in 0.2 M KOH in methanol followed by passage through a Dowex 50 (HI form) column (29). The concentration (nanomoles) of each LTA preparation was estimated from the phosphorus content (1) on the basis of an average 27.5 nmol of P/nmol of LTA (31). Dipalmitin (Sigma Chemical Co., St. Louis, Mo.), dipalmitoyl phosphatidyl glycerol (Serdary Research Labs, London, Ontario, Canada), monogalactosyl diglyceride (Analabs, Inc., North Haven, Conn.) and beef heart cardiolipin (Applied Science Labs, State College, Pa.) were dissolved in ethanol at concentrations of 5 to 7.5 mg/ml. Cardiolipin was deacylated in methanolic KOH as described above for LTA.
RESULTS Gel filtration of [3H]glycerol-labeled LTA of S. faecalis. The two successive extracts (hot water and hot aqueous phenol) of the cell residue each showed three peaks of 3H-containing material (Fig. 1). The first peak (peak I) eluted near the void volume of the column, as expected for fully acylated micellar LTA. The second peak (peak II) was included in the column and has a molecular size consistent with deacylated or partially deacylated (nonmicellar) LTA (9). The chemical nature of the 3H-labeled material in the third peak (peak III), which elutes close
194
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CLEVELAND ET AL.
500 200 100
x
45
0-
030
-
15
-
I
30
40
50 60 70 80 FRACTION NUMBER
90
FIG. 1. Elution profiles of[3H]glycerol on a Sepharose 6B and Bio-Gel P30 column system of hot-water and hot-aqueous-phenol extracts ofS. faecalis 9790. Counts are expressed as disintegrations per minute, and each profile reflects the total disintegrations per minute in the extract.
to the total volume of the double-column system, is not known. The hot-water extract contained a substantial level of peak II material (about 50% of total material), a lesser amount of peak III material (about 30%), and a relatively small amount of peak I material (about 20%). In contrast, the hot-aqueous-phenol extract contained very large amounts of peak I material (95% or more of total material) and only traces of peaks II and III. The total amount of 3H recovered in the aqueous phenol extract was about 10 times that in the hot-water extract. LTA extracted from S. faecalis 9790 with hot aqueous phenol has previously been shown to react with antibody specific for polyglycerol phosphate (10). Material from peaks I and II from the Sepharose 6B column has been shown to strongly react with streptococcal group D (kojibiose-specific antiserum [91). In addition, acid hydrolysis and paper chromatography of unfractionated LTA and samples of peak I and peak II from S. faecalis 9790 have shown the presence of [3H]glycerol-containing substances with the same mobility as glycerol, glycerol monophosphate, and glycerol diphosphate (9, 10).
Experiments performed using ['4C]acetate in addition to [3H]glycerol showed significant 14C in peak I but not in peak II (data not shown), which is consistent with the identification of peak I as acylated LTA and peak II as deacylated LTA. Effects of peaks I and II on wall lysis. The pooled fractions from peaks I and II (from hotaqueous-phenol extracts) were tested for their effect on lysis of cell walls. Material from peak I inhibited wall lysis at very low concentrations (Fig. 2). Only 36 nmol of phosphorus per mg of wall (equivalent to 1.3 nmol of LTA/mg of wall) was sufficient to give the maximum inhibition observed (70%). This ratio of LTA to wall is below the ratio of LTA to wall (3 to 5 nmol/mg) calculated to be present in cells of S. faecalis (3). Furthermore, we have found that in the same lysis test system peak I material is comparable in specific inhibitory activity to purified LTA from L. fermentum 6991, which has been chemically and serologically characterized (12). Neither peak II nor chemically deacylated peak I showed significant inhibition at concentrations up to four times the concentration of peak I that gave maximal inhibition. A much higher concentration of peak II gave some inhi-
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LTA INHIBITION OF WALL AUTOLYSIS
100 w
A):
0
o
Ao0
80 0
D 60
\* Peak I 0 Deacylated Peak I a
Peak 11
-J
0 c:-
_ t t* z 40 t 0 0 z
C 20 w
0.
0
I
l 20 3. 0l 1.0l.o 2.0 3.0
4.0 4.0
LTA (nmoles/mg wall) FIG 2. Effect of fully acylated (peak I), partially deacyllated (peak II), and chemically deacylated LTA from 'S. faecalis on autolysis of walls from S. faecalis. Peaks I and n were obtained from hot-aqueousphenoo1 extracts. Percent control rate is based on control rc:Pte constants of 0.2 to 0.6/h.
195
terial in peak III was partially (or slowly) dialyzable. Thus, it was not easily possible to remove salts from this fraction to quantitatively examine inhibitory activity in peak III in the absence of artifacts. Effects of lipids on wall lysis. A glycerol label in S. faecalis appears to be restricted primarily to lipids and LTA (13). Several observations, such as the loss of inhibitory activity upon deacylation of LTA and a suggestion of slight inhibitory activity in peak III (data not shown), led us to examine the effects of various lipids on lysis of the wall-enzyme complex. Both commercially available, highly purified lipid and lipid fractions extracted from S. faecalis were tested. Beef heart cardiolipin was a surprisingly effective inhibitor of wall lysis (Fig. 3). Substantial levels of inhibition, reaching a maximum of 80%, were attained at 10 to 20 nmol per mg of wall.
A comparison of the data shQwn in Fig. 2 and 3 shows that 55% inhibition was observed with either about 2 nmol of beef heart cardiolipin or about 1 nmol of peak I LTA per mg (dry weight) of wall. These observations suggest that LTA in ak I is abou pe autolysist as is andinhibitorDe-of wall is beefeffect heart cardiolipin.
bition (about 50% inhibition at 23.5 nmol/mg of wall). In contrast, high concentrations of chemically deacylated peak II tended to stimulate 100 wall lysis to a highly variable extent. For example, in one experiment 15.4 nmol of chemi- w cally deacylated LTA/mg of wall stimulated wall lysis by about 2.5-fold, whereas in another cexperiment (3.9 nmol/mg wall) lysis was stimu- o 80 lated by 16%. Hot-water extracts have been previously re- 0-J ported to inhibit autolysis in S. faecalis when added in amounts that were large as compared D 60 -J to the amounts used in the experiments re- 0 ported here (M. Sayare, L. Daneo-Moore, and G. D. Shockman, Abstr. Annu. Meet. Am. Z 40 Soc. Microbiol. 1972, G106, p. 48). Variable de- 0 grees of inhibition of wall lysis were obtained from unfractionated hot-water extracts in the wz experiments reported here. This was appar- c 20 ently due, at least in part, to the relatively low 0.w amounts of peak I material recovered in hotwater extracts. Peak III material from hot-water extracts was also tested for inhibitory activity. Assays of large amounts of partially dialyzed peak III material indicated the presence of a low level of inhibitory activity. However, fractionation of a hot-water extract of a culture grown in the presence of [3H]glycerol and ['4C]acetate showed that peak III contained more than one component (data not shown). Furthermore, ma-
0
0
20 10 30 40 PHOSPHOLIPID (nmoles/mg wall) FIG. 3. Effect ofbeef heart cardiolipin (acylated or chemically deacylated) and of various lipid fractions (from S. faecalis) on autolysis of walls of S. faecalis. PG, Phosphatidyl glycerol; DiPG, diphosphatidyl
glycerol (cardiolipin); aaPG, aminoacyl phosphatidyl glycerol.
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lated) LTA into the growth medium (9, 12), inhibition obtained is not much lower than that obtained with peak I LTA. As is the case for some other biological activities of LTA (31), deacylation removed inhibition of wall autolysis. Deacylated beef heart cardiolipin also was without inhibitory activity. The observation that inhibitory activity was lost after deacylation is of potential physiological significance, since it was shown recently that several microorganisms excrete deacylated or partially deacylated (as well as fully acylated) LTA into the growth medium (9, 12), whereas intact cells contain a predominance of acylated LTA (Fig. 1). The mechanism(s) of action of the inhibition reported here remains to be elucidated. It would be of interest to determine whether the inhibition is exerted on the wall substrate or on the enzyme. Also unclear at this time is the relationship between the inhibition obtained with LTA and that observed in the presence of certain lipids. However, the structural similarity between cardiolipin and LTA has been shown by experiments in which deacylated cardiolipin inhibits, to a variable extent, the reaction of LTA with antibodies specific to the polyglycerol phosphate backbone of various LTA preparations (30). In S. faecalis, LTA accounts for about 1 to 2% of the cellular dry weight (11), whereas phospholipids account for about 3.5% of the cellular dry weight (5, 22). Assuming an average of 25 to 30 phosphate residues per LTA and an average of 1 to 2 phosphate residues per lipid, respectively, it would appear that, per unit cellular dry weight, there is about two to three times more inhibitory potential from phospholipid TABLE 1. Effect of lipids from various sources on than from LTA. However, the high level oflipid wall lysis phosphorus in cells does not necessarily rule Con- out LTA as a regulatory molecule, especially o auto- since LTA can be excreted in both acylated and Lipid allwal trol wg/ *gm lytic rate deacylated forms (9). Removal of inhibitory activity by deacylation 92 20.9 1,2-Dipalmitin could provide a mechanism for the control of 42.1 86 autolytic activity, e.g., by action of a deacyl79.4 79 ase. It would not be unreasonable to assume 52 19.8 Dipalmitoyl phosphatidyl that deacylated LTA, which might no longer be glycerol 39.1 48 of use to the cell, would be excreted. In addi78.2 34 tion, the location of LTA on the outer surface of the protoplast membrane (10, 11), from which it 14.0 93 Monogalactosyl diglyceride is easily released and perhaps deacylated, 27.0 89 would facilitate interaction between this mole51.5 80 cule and a native autolysin functioning in adjacent areas of wall. Deacylation would prevent 132 94.9 Glycolipids (S. faecalis 9790) 36 lipophilic interactions between the lipid moiety Neutral lipids (S. faecalis 94.9 9790) of LTA and cellular membrane components 60 96.2 Phospholipids (S. faecalis that undoubtedly would be important in main9790) taining this configuration.
acylated beef heart cardiolipin at concentrations of 45 nmol/mg (dry weight) of wall failed to inhibit (or stimulate) wall autolysis. Dipalmitoyl phosphatidyl glycerol also inhibited wall lysis (Table 1), whereas 1,2-dipalmitin and a monogalactosyl diglyceride did not have significant inhibitory activity. For the last two compounds, the maximal levels of inhibition observed were significantly lower than those obtained with either phosphatidyl glycerol (Fig. 3) or cardiolipin (Table 1). It seems possible that the maximal achievable levels of inhibition were limited by the solubility of various lipids in the aqueous assay system used. A crude phospholipid fraction from S. faecalis inhibited wall autolysis (Table 1), as did the cardiolipin, phosphatidyl glycerol, and aminoacyl phosphatidyl glycerol fractions purified from the crude phospholipid fraction of S. faecalis (Fig. 3). The neutral lipid fraction of S. faecalis was also inhibitory (Table 1). In contrast, the crude glycolipid fraction obtained from S. faecalis was slightly stimulatory. DISCUSSION The present study demonstrates that LTA isolated from S. faecalis '9790 inhibited the muramidase activity of homologous organisms. The muramidase of S. faecalis was also inhibited by commercially obtainable lipids and by several of the lipids and phospholipids that are commonly found in bacteria. Interpretation of the quantitative data is limited by the solubility of various lipid molecules in the assay system. Nevertheless, it would appear that, at least for the phosphatidyl glycerol and cardiolipin fractions, the order of magnitude of the
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Studies of the kinetics of inhibition by both LTA and lipids as well as determination of the accumulation of each in various physiologically altered states are required. It is also entirely possible that more than one cellular constituent acts as a regulatory molecule of the muramidase of S. faecalis. In S. faecalis a single enzyme activity (21) is present to perform all of the physiological roles that have been assigned to bacterial autolysins (15, 19). Some of these roles may depend on autolysins located well within the wall, whereas others may require enzymatic action at the outer boundary of the wall. ACKNOWLEDGMENTS This work was supported by Public Health Service grant AI 05044 from the National Institute of Allergy and Infectious Diseases and grant GB 20813 from the National Science Foundation. A. J. W. was a senior foreign Fellow of the American Association of Dental Research. R. C. was supported by Public Health Service training grant CA 05280 from the National Cancer Institute. Excellent technical assistance from T. Greber is acknowledged. We thank B. Rosan for the use of his Ribi cell fractionator. LITERATURE CITED 1. Ames, B. N., and D. T. Dubin. 1960. The role of polyamines in the neutralization of bacteriophage deoxyribonucleic acid. J. Biol. Chem. 235:769. 2. Briles, E. B., and A. Tomasz. 1973. Pneumococcal Forssman antigen. A choline-containing lipoteichoic acid. J. Biol. Chem. 248:639.-6397. 3. Cleveland, R. F., J.-V. Holtje, A. J. Wicken, A. Tomasz, L. Daneo-Moore, and G. D. Shockman. 1975. Inhibition of bacterial wall lysins by lipoteichoic acids and related compounds. Biochem. Biophys. Res. Commun. 67:1128-1135. 4. Forsberg, C. W., P. B. Wyrick, J. B. Ward, and H. J. Rogers. 1973. Effect of phosphate limitation on the
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morphology and wall composition ofBacillus licheniformis and its phosphoglucomutase-deficient mutants. J. Bacteriol. 113:969-984. Ganfield, M.-C. W., and R. A. Pieringer. 1975. Phosphatidylkojibiosyl diglyceride. The covalently linked lipid constituent of the membrane lipoteichoic acid from Streptococcus faecalis (faecium) ATCC 9790. J. Biol. Chem. 250:702-709. Goebel, W. F., T. Shedlovsky, G. I. Lavin, and M. H. Adams. 1943. The heterophile antigen of pneumococcus. J. Biol. Chem. 148:1-15. Hartmann, R., J.-V. Holtje, and U. Schwarz. 1972. Targets of penicillin action in Escherichia coli. Nature (London) 235:426-429. Holtje, J.-V., and A. Tomasz. 1975. Lipoteichoic acid: a specific inhibitor of autolysin activity in pneumococcus. Proc. Natl. Acad. Sci. U.S.A. 72:1690-1694. Joseph, R., and G. D. Shockman. 1975. The synthesis and excretion of glycerol teichoic acid during growth of two streptococcal species. Infect. Immun. 12:333-
338. 10. Joseph, R., and G. D. Shockman. 1975. Cellular localization of lipoteichoic acid in Streptococcus faecalis. J. Bacteriol. 122:1375-1386. 11. Knox, K. W., and A. J. Wicken. 1973. Immunological properties of teichoic acids. Bacteriol. Rev. 37:215257.
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12. Markham, J. L., K. W. Knox, A. J. Wicken, and M. J. Hewett. 1975. Formation of extracellular lipoteichoic acid by oral streptococci and lactobacilli. Infect. Immun. 12:378-386. 13. Pieringer, R. A., and R. T. Ambron. 1973. A method for the specific labeling of the glycerol in glyceride-containing lipids of Streptococcus faecalis ATCC 9790. J. Lipid Res. 14:370-372. 14. Pooley, H. M., and G. D. Shockman. 1970. Relationship between the location of autolysin, cell wall synthesis, and the development of resistance to cellular autolysis in Streptococcus faecalis after inhibition of protein synthesis. J. Bacteriol. 103:457-466. 15. Rogers, H. J. 1970. Bacterial growth and the cell envelope. Bacteriol. Rev. 34:194-214. 16. Rogers, H. J., and C. W. Forsberg. 1971. Role of autolysins in the killing of bacteria by some bacterial antibiotics. J. Bacteriol. 108:1235-1243. 17. Sayare, M., L. Daneo-Moore, and G. D. Shockman. 1972. Influence of macromolecular biosynthesis on cellular autolysis in Streptococcus faecalis. J. Bacteriol. 112:337-344. 18. Shockman, G. D. 1963. Amino acids, p. 567-673. In F. Kavanagh (ed.), Analytical microbiology. Academic Press Inc., New York. 19. Shockman, G. D., L. Daneo-Moore, and M. L. Higgins. 1974. Problems of cell wall and membrane growth, enlargement and division. Ann. N.Y. Acad. Sci. 235:161-197. 20. Shockman, G. D., H. M. Pooley, and J. S. Thompson. 1967. Autolytic enzyme system of Streptococcus faecalis. III. Localization of the autolysin at the sites of cell wall synthesis. J. Bacteriol. 94:1525-1530. 21. Shockman, G. D., J. S. Thompson, and M. J. Conover. 1967. The autolytic enzyme system of Streptococcus faecalis. II. Partial characterization of the autolysin and its substrate. Biochemistry 6:1054-1065. 22. Toennies, G., G. D. Shockman, and J. J. Kolb. 1963. Differential effects of amino acid deficiencies on bacterial cytochemistry. Biochemistry 2:294-296. 23. Tomasz, A., A. Albino, and E. Zaneti. 1970. Multiple antibiotic resistance in a bacterium with suppressed autolytic system. Nature (London) 227:138-140. 24. Toon, P., P. E. Brown, and J. Baddiley. 1972. The lipid-teichoic acid complex in the cytoplasmic membrane of Streptococcus faecalis N.C.I.B. 8191. Biochem. J. 127:399-409. 25. Vorbeck, M. L., and G. V. Marinetti. 1965. Intracellular distribution and characterization of the lipids of Streptococcus faecalis (ATCC 9790). Biochemistry 4:296-305. 26. Vorbeck, M. L., and G. V. Marinetti. 1965. Separation of glycosyl diglycerides from phosphatides using silicic acid column chromatography. J. Lipid Res. 6:36. 27. Wells, M. A., and J. C. Dittmer. 1963. The use of Sephadex for the removal of nonlipid contaminants from lipid extracts. Biochemistry 2:1259-1263. 28. Wicken, A. J., J. W. Gibbens, and K. W. Knox. 1973. Comparative studies on the isolation of membrane lipoteichoic acid from Lactobacillus fermenti. J. Bacteriol. 113:365-372. 29. Wicken, A. J., K. W. Knox. 1970. Studies on the group F antigen of lactobacilli: isolation of a teichoic acidlipid complex from Lactobacillus fermenti NCTC 6991. J. Gen. Microbiol. 60:293-301. 30. Wicken, A. J., and K. W. Knox. 1971. A serological comparison of the membrane teichoic acids from lactobacilli of different serological groups. J. Gen. Microbiol. 67:251-254. 31. Wicken, A. J., and K. W. Knox. 1975. Lipoteichoic acids: a new class of bacterial antigen. Science 187:1161-1167.
