JouRNuL OF BACTRIOLOGY, Mar. 1979, p. 1176-1179 0021-9193/79/03-1176/04$02.00/0
Vol. 137, No. 3
Enzymatic Deacylation of Lipoteichoic Acid by Protoplasts of Streptococcus faecium (Streptococcus faecalis ATCC 9790) ROBERT E. KESSLERt AND GERALD D. SHOCKMAN* Department of Microbiology and Immunology, Temple University Health Sciences Center, Philadelphia, Pennsylvania 19140 Received for publication 26 October 1978
High-molecular-weight, micellar lipoteichoic acid (LTA) was converted to a lower-molecular-weight, apparently deacylated polymer when the forner was incubated in the presence of growing protoplasts of Streptococcus faecium (S.
faecalis ATCC 9790), but not when incubated in fresh or spent protoplast medium. The mobility of the low-molecular-weight polymer upon agarose gel electrophoresis was indistinguishable from that of native extracellular lipoteichoic acid (LTAX) from this organism or from chemically deacylated LTA. Native LTAx was shown to contain less than one fatty acid equivalent per 18 LTAx molecules, in contrast to the 4:1 ratio of fatty acids to polyglycerolphosphate chains in micellar LTA. Lipoteichoic acids have been found both intracellularly (LTAJ) (2, 3, 7, 9, 11, 14, 16, 17) and extraceliularly (LTAX) (5, 6, 8). LTAi appears to be located on the outer portion of the cytoplasmic membrane (15). Both LTA; and LTAX may be extracted and fractionated as a high-molecular-weight micellar aggregate, as a low-molecular-weight form, or as mixtures of these two forms (5, 6, 8). Although the low-molecularweight form apparently contains low levels of fatty acids compared to micellar LTA (8), it has similar physical properties to micellar LTA that has been chemically deacylated (2; unpublished data). Chemical deacylation during extraction may account for the presence of this low-molecular form in cellular extracts. However, it has also been isolated from extracellular culture fluids under conditions that do not result in deacylation of micellar LTA (5,6,8). In addition, the LTAX from Streptococcus faecium (S. faecalis ATCC 9790) is entirely of the low-molecular-weight form after isolation under non-deacylating conditions (5, 6). Recently, evidence was presented that low-molecular-weight LTA. was entirely derived from LTAi (6). These observations suggest the presence of an enzymatic activity capable of converting high-molecular-weight, acylated LTA to the low-molecular-weight form in this species. Evidence for such an activity is presented here. Furthermore, using a very sensitive dual radioactive labeling technique, we also show that the residual fatty acid content of native LTAX is extremely low in comparison to micellar LTAi.
MATERIALS AND METHODS Culture conditions and measurement. S. facecium (S. faecalis ATCC 9790) was grown in a chemically defined medium (FCM) modified from that of Toennies and Gallant (13). In certain experiments the formulation of this medium was further modified to contain 0.5 mg of sodium acetate per ml rather than the usual 6 mg/ml when [1-14C]acetic acid, sodium salt (Amersham Searle, 58.1 mCi/mmol) was included (4). Lower concentrations of sodium acetate were not used because autolysis has been observed in cultures grown in concentrations of acetate less than 0.3 mg/ml (15). In dual labeling experiments, cultures were grown in the presence of 2 ,uCi of [1_-4C]acetic acid per ml and 1 lsCi of [2-3H]glycerol (Amersham Searle, 202 mCi/ mmol) per ml. Methods of media preparation, culture conditions, and measurement have been described previously (6). Protoplasts were prepared by the method of Roth et al. (10), and each preparation was observed microscopically to confirm protoplast formation. Protoplasts or cells grown in the presence of LTA were harvested by centrifuging 1.2-ml samples (12,000 x g, 5 min, 4°C) in conical polypropylene tubes with a Brinkman 3200 centrifuge. The uppermost 1.0 ml of
supematant medium was then carefully removed and dialyzed before lyophilization. LTA extraction, purification, and quantitation. Procedures for the isolation and quantitation of LTA were as previously described (6, 12). The terms peak I and peak II denote acylated, miceliar LTA and low-molecular-weight, apparently deacylated LTA, respectively (6, 12). The [3H]glycerol-labeled, micellar LTA used in the protoplast and whole cell incubation experiments was prepared (6, 12) from ca. 1.5 mg (dry weight) of cells grown in medium containing 20 iLCi of [2-3H]glycerol per ml and mixed with 4 mg of unlabeled LTA. By assuming that LTA constituted 1% of the dry weight of the labeled cells (7), we calculated t Present address: The Rockefeller University, New York, that the dry weight of the labeled LTA would be ca. NY 10021. 0.4% of the dry weight of the unlabeled LTA and thus 1176
DEACYLATION OF LIPOTEICHOIC ACID
VOL. 137, 1979
with 18 polyglycerolphosphate chains of LTAX. Thus, LTAX and, by inference, peak II LTA1 appeared to be deacylated; i.e., contain very few, if any, polyglycerolphosphate molecules with intact fatty acid(s). In similar experiments where some peak II (ca. 5% of total LTAJ) was found in aqueous phenol extracts of whole cells, the level of [1-'4C]acetate was at or below the background (data not shown). The extracellular material in the peak of very low electrophoretic mobility (peak "O", Fig. 1B) that contains [1'C]acetate but not [3H]glycerol has not yet been identified. It could represent extracellular lipid although it lacks glycerol, and excretion or turnover of lipid or free fatty acids was not noted in this organism in previous studies (6). Demonstration of an enzymatic activity for conversion of high-molecular-weight LTA to its low-molecular-weight form. To look for the presence of an appropriate enzymatic activity capable of converting acylated LTA to the apparently unacylated, low-molecular-weight form, radiolabeled acylated LTA was added to and incubated with growing cultures of intact streptococci and protoplasts. An LTA preparation was added to a final concentration of 100 ,ug/ml to cultures of intact streptococci, cultures of protoplasts, and to medium in which protoplasts had grown for 2 h. After incubation at 370C for 2 h, protoplasts and cells were removed by centrifugation. Samples (2.0 ml) of supernatant medium were dialyzed, lyophilized, and then resuspended in 100 ul of electrophoresis buffer. Ca. 95 and 93% of the added LTA was recovered in the supernatant from protoplasts and cells, respectively. Of this, in the case of LTA recovered from protoplast
its contribution to the total dry weight was considered to be negligible. The mixture was rechromatographed on the Sepharose 6B:Bio-Gel P-30 column system. A final specific activity of 6.25 x 105 dpm/mg of LTA was calculated. LTA with the electrophoretic mobility of peak II accounted for less than 0.1% of the total LTA as determined by agarose electrophoresis. This preparation was used to detect enzymatic activity capable of converting acylated LTA to its low-molecular-weight form. Radioactivity of samples was determined as described previously (6).
RESULTS Estimation of fatty acid content of LTA1 and LTAX. The result of agarose gel electrophoresis of an aqueous phenol extract of whole streptococci grown in the presence of [2-3H]glycerol and [1-'4Clacetate is shown in Fig. 1A. The ratio of 3H to C was 9:1 in peak I LTA. There was insufficient peak II to calculate a ratio for the radioactive material contained within this peak. Gel electrophoresis of concentrated, dialyzed spent culture medium showed a symmetrical [2-3H]glycerol profile at the position of peak II, as previously described for LTAX from this organism (6, 12). The gel slice representing the tip of the peak contained 6,400 dpm of 3H, but a mean of 30 dpm (0 to 10 dpm above the background) of 14C was counted in each slice under the 3H peak. If LTAx contained only a single fatty acid per molecule, in contrast to the four fatty acids covalently linked to each molecule of LTAi, then a minimum of 180 dpm of [114C]acetate would have been found in the same peak gel slice. (If more than one fatty acid remained attached to LTA., correspondingly greater counts would have been found.) This data has been interpreted as indicating that less than one fatty acid equivalent was associated -12
A.
*
0 12 B. 11 II .J* X0
010
2 0
4
6
~~~16
E-8
-
10 -8
-j 6
~~~~~~~~~~~E cl 122J 6_j
04 -I
8
DITAC FRO
0
1177
2
4
ORGI
6
8
LIL
4-
lam 0~~~0DITNEFO
10
DISTANCE FROM ORIGIN (cm)
0
2
4
RGN(m
6
8
10
DISTANCE FROM ORIGIN (cm)
FIG. 1. Agarose gel electrophoresis of LTAi (A) and LTAX (B) from a culture grown in the presence of [23H]glycerol and [1-'4C]acetate. Sample preparation and electrophoresis was carried out as previously described (6, 12). The two peak areas have been labeled I and II, and correspond to the positions of acylated micellar LTA and low-molecular-weight, apparently deacylated LTA, respectively (6, 12).
1178
KESSLER AND SHOCKMAN
J. BACTERIOL.
supernatants, ca. 15% was deacylated. The amounts of deacylated LTA in 20-p1 samples were calculated from the radioactivity contained in peak II (as defined in Fig. 1) after agarose electrophoresis, assuming an approximate mo0 E lecular weight of 10,000 for LTA. From electrophoresis of untreated samples, values of peak II 3 below 0.025 nmol/ml were calculated to be below the limits of detection and reported as not detectable. Very little deacylated LTA was de02 tected after 2 h of incubation with growing intact cells or with spent protoplast medium, but a substantial amount of deacylated LTA was de0 tected after 2 h of incubation with growing protoplasts (Table 1). The small amount of peak II found after incubation with spent medium may 90 60 120 30 0 have been due to contamination with a small TIME (minutes) number of protoplasts or a result of the small FIG. 2. Kinetics of conversion of exogenous LTA degree of lysis of the original protoplasts that Protoplasts of had been grown in this medium as previously to deacylated monomer by protoplasts. faecium were resuspended in protoplast medium reported for this system (10). The kinetics of S. 200 ug of [3H]LTA per ml. At the times conversion of exogenous LTA to the deacylated containing were removed and processed as samples indicated, monomer by growing protoplasts is shown in detailed in Materials and Methods. The amount of Fig. 2. LTA (200 ,ug/ml) was added to growing deacylated LTA is expressed as nmoles per 2 ml of protoplasts, and samples were removed at the the original culture. times shown and processed as described above. DISCUSSION Recovery of total LTA (acylated and deacylated) from the supernatants in these experiThe existence of an enzymatic activity for the conversion of micellar LTA (peak I) to unacyments was ca. 90%. lated LTA (peak II) has been demonstrated. By TABLE 1. Effect of incubation of LTA with growing exam ning the surrounding culture medium after incubation of peak I with growing protoplasts S. faecium cells, growing protoplats, and spent protoplast mediuma rather than extracting the protoplasts themselves, we were able to avoid the risk of chemical DeacylCulture turated hydrolysis that may accompany any extraction LTAC bidity Sample procedure. The enzyme appears to be cell bound (nmol/ (AOD)b because very little peak II could be detected ml) after incubation of peak I in spent protoplast 50 700 0.05 Cell supernatant medium. Contamination with a small number of 100 450 0.55 Protoplast supernatant or a small degree of lysis (10) may protoplasts 0.05 Spent protoplast medium account for the appearance of small amounts of NDd Uninoculated protoplast mepeak II in spent protoplast medium samples. dium Location of the enzyme on the outer surface of LTA was added to a final concentration of 100 pg/ the membrane is likely, considering the rhemml to growing intact cells, growing protoplasts, unin- brane location of LTAi (7, 15, 17). In the experoculated protoplast medium, and medium in which fatty acids of protoplasts had grown for 2 h but had been removed imental situation described, the by centrifugation. Each was incubated at 37°C for 2 h. the hydrophobic lipid moiety are presumed to Cells and protoplasts were separated from medium by be adsorbed to the exposed protoplast memcentrifugation (12,000 x g, 10 min, 4°C). The super- brane in a manner analagous to that of adsorpnatant media were dialyzed (2 ml per sample), lyoph- tion of LTA to erythrocytes (7, 9, 17). A memilized, and resuspended in 100 id of electrophoresis brane-bound enzyme then "deacylates" the buffer. A 20-pl amount of each was subjected to elec- LTA, releasing the very hydrophilic portion to trophoresis (see Fig. 2). the surrounding medium. The very small b AOD, Adjusted optical density. of peak II found after incubation of peak Calculation based on the dpm contained under amount in the I presence of growing cells may have peak II, a specific activity of 6.25 x 105 dpm/mg and resulted from restriction of access to the memmolecular weight of 10,000. d ND, Not detectable. Lower limit of detection was brane by the intact cell wall. In vivo, LTAi is a normal membrane component and thus, would 0.025 nmol/ml (see text). a
DEACYLATION OF LIPOTEICHOIC ACID
VOL. 137, 1979
be readily accessible to such an enzyme. The exact cleavage site(s) was (were) not determined in the present study. Thus, characterization of the enzymatic action as a true deacylation cannot be made. Cleavage at some other point(s) within the lipid moiety or between it and the polyglycerolphosphate chain may be responsible for the observed effects. Clarification of this point must await a complete structural analysis on the material found at the position of peak II after electrophoresis of LTA exposed to growing protoplasts. However, this material (i) elutes from Sepharose 6B columns at the volume expected for chemically deacylated LTA and for LTAX, (ii) contains glycerol and glycerol phosphate (5), and (iii) migrates to a position previously shown by agarose gel electrophoresis of labeled LTA and by crossed immuno- and affinoelectrophoresis to be characteristic of chemically deacylated LTA as well as native LTAX (6, 12). Furthermore, native LTAX was shown to contain little, if any, ['4C]acetate (Fig. 1B)-an indicator of the presence of fatty acids in micellar LTA;. Finally, all incorporated radioactive glycerol and 90% of incorporated radioactive acetate can be accounted for in lipid and LTA (6). Therefore, at the very least, enzymatic activity for conversion of acylated LTA to a molecule with electrophoretic properties of native LTA, from this organism has been demonstrated. The fact that only deacylated LTA has been found as LTAK, in contrast to that from other members of the Lactobacillaceae (5,8), suggests that two mechanisms, only one of them operating in S. faecium, may exist for the excretion of LTA. It may be that in a bacterium with very low or no loss of envelope components to the surrounding medium such as this particular strain (1, 6), LTA with intact fatty acids is not found extracellularly. Excretion preceded by deacylation may have a very different physiological function compared to loss of intact LTA. ACKNOWLEDGMENTS This research was supported by Public Health Service research grants Al 05044 from the National Institute of Allergy and Infectious Diseases and DE 03487 from the National Institute of Dental Research. We thank A. J. Wicken for many helpful suggestions during these studies.
1179
LITERATURE CITED 1. Boothby, D., L. D. Moore, M. L. Higgins, J. Coyette, and G. D. Shockman. 1973. Turnover of bacterial cell wall peptidoglycans. J. Biol. Chem. 248:2161-2169. 2. Button, D., and N. L. Hemmings. 1975. Teichoic acids and lipids associated with the membrane of a Bacillus licheniformis mutant and the membrane lipids of the parental strain. J. Bacteriol. 128:149-156. 3. Coley, J., M.. Duckworth, and J. Baddiley. 1972. The occurrence of lipoteichoic acids in the membranes of Gram-positive bacteria. J. Gen. Microbiol. 73:587-591. 4. Dezelee, P., and G. D. Shockman. 1975. Studies of the formation of peptide cross-links in the cell wall peptidoglycan of Streptococcus faecalis. J. Biol. Chem. 250: 6806-6816. 5. Joseph, R., and G. D. Shockman. 1975. Synthesis and excretion of glycerol teichoic acid during growth of two streptococcal species. Infect. Immun. 12:333-338. 6. Kessler, R. E., and G. D. Shockman. 1979. Precursorproduct relationship of intracellular and extracellular lipoteichoic acids of Streptococcus faecium. J. Bacteriol. 137:869-877. 7. Knox, K. W., and A. J. Wicken. 1973. Immunological properties of teichoic acids. Bacteriol. Rev. 37:215-257. 8. 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. 9. Ofek, I., E. H. Beachey, W. Jefferson, and G. L. Campbell. 1975. Cell membrane-binding properties of group A streptococcal lipoteichoic acid. J. Exp. Med. 141:990-1003. 10. Roth, G. S., G. D. Shockman, and L. Daneo-Moore. 1971. Balanced macromolecular biosynthesis in "protoplasts" of Streptococcus faecalis. J. Bacteriol. 105: 710-717. 11. Rudczynski, A. B., and R. W. Jackson. 1978. The properties of a lipoteichoic acid antigen from Streptococcuspyogenes. Immunochemistry 15:83-91. 12. Shockman, G. D., R. Kessler, J. B. Cornett, and M. Mychajlonka. 1978. Turnover and excretion of streptococcal surface components. Adv. Exp. Med. Biol. 107: 803-814. 13. Toennies, G., and D. L. Gallant. 1949. The relationship between photometric turbidity and bacterial concentration. Growth 13:7-20. 14. Toon, P., P. E. Brown, and J. Baddiley. 1972. The lipid-teichoic acid complex in the cytoplasmic membrane of Streptococcus faecalis NCIB 8191. Biochem. J. 127:399-409. 15. Van Driel, D., A. J. Wicken, M. R. Dickson, and K. W. Knox. 1973. Cellular location of the lipoteichoic acids of Lactobacillus fermnenti NCTC 6991 and Lactobacillus casei NCTC 6375. J. Ultrastructure Res. 43: 483497. 16. Wicken, A. J. and K. W. Knox. 1970. Studies on the Group F antigen of lactobacilli: isolation of a teichoic acid-lipid complex from Lactobacillus fermenti NCTC 6991. J. Gen. Microbiol. 60:293-301. 17. Wicken, A. J., and K. W. Knox. 1975. Lipoteichoic acids: a new class of bacterial antigen. Science 187: 1161-1167.
Vol. 137, No. 3
Enzymatic Deacylation of Lipoteichoic Acid by Protoplasts of Streptococcus faecium (Streptococcus faecalis ATCC 9790) ROBERT E. KESSLERt AND GERALD D. SHOCKMAN* Department of Microbiology and Immunology, Temple University Health Sciences Center, Philadelphia, Pennsylvania 19140 Received for publication 26 October 1978
High-molecular-weight, micellar lipoteichoic acid (LTA) was converted to a lower-molecular-weight, apparently deacylated polymer when the forner was incubated in the presence of growing protoplasts of Streptococcus faecium (S.
faecalis ATCC 9790), but not when incubated in fresh or spent protoplast medium. The mobility of the low-molecular-weight polymer upon agarose gel electrophoresis was indistinguishable from that of native extracellular lipoteichoic acid (LTAX) from this organism or from chemically deacylated LTA. Native LTAx was shown to contain less than one fatty acid equivalent per 18 LTAx molecules, in contrast to the 4:1 ratio of fatty acids to polyglycerolphosphate chains in micellar LTA. Lipoteichoic acids have been found both intracellularly (LTAJ) (2, 3, 7, 9, 11, 14, 16, 17) and extraceliularly (LTAX) (5, 6, 8). LTAi appears to be located on the outer portion of the cytoplasmic membrane (15). Both LTA; and LTAX may be extracted and fractionated as a high-molecular-weight micellar aggregate, as a low-molecular-weight form, or as mixtures of these two forms (5, 6, 8). Although the low-molecularweight form apparently contains low levels of fatty acids compared to micellar LTA (8), it has similar physical properties to micellar LTA that has been chemically deacylated (2; unpublished data). Chemical deacylation during extraction may account for the presence of this low-molecular form in cellular extracts. However, it has also been isolated from extracellular culture fluids under conditions that do not result in deacylation of micellar LTA (5,6,8). In addition, the LTAX from Streptococcus faecium (S. faecalis ATCC 9790) is entirely of the low-molecular-weight form after isolation under non-deacylating conditions (5, 6). Recently, evidence was presented that low-molecular-weight LTA. was entirely derived from LTAi (6). These observations suggest the presence of an enzymatic activity capable of converting high-molecular-weight, acylated LTA to the low-molecular-weight form in this species. Evidence for such an activity is presented here. Furthermore, using a very sensitive dual radioactive labeling technique, we also show that the residual fatty acid content of native LTAX is extremely low in comparison to micellar LTAi.
MATERIALS AND METHODS Culture conditions and measurement. S. facecium (S. faecalis ATCC 9790) was grown in a chemically defined medium (FCM) modified from that of Toennies and Gallant (13). In certain experiments the formulation of this medium was further modified to contain 0.5 mg of sodium acetate per ml rather than the usual 6 mg/ml when [1-14C]acetic acid, sodium salt (Amersham Searle, 58.1 mCi/mmol) was included (4). Lower concentrations of sodium acetate were not used because autolysis has been observed in cultures grown in concentrations of acetate less than 0.3 mg/ml (15). In dual labeling experiments, cultures were grown in the presence of 2 ,uCi of [1_-4C]acetic acid per ml and 1 lsCi of [2-3H]glycerol (Amersham Searle, 202 mCi/ mmol) per ml. Methods of media preparation, culture conditions, and measurement have been described previously (6). Protoplasts were prepared by the method of Roth et al. (10), and each preparation was observed microscopically to confirm protoplast formation. Protoplasts or cells grown in the presence of LTA were harvested by centrifuging 1.2-ml samples (12,000 x g, 5 min, 4°C) in conical polypropylene tubes with a Brinkman 3200 centrifuge. The uppermost 1.0 ml of
supematant medium was then carefully removed and dialyzed before lyophilization. LTA extraction, purification, and quantitation. Procedures for the isolation and quantitation of LTA were as previously described (6, 12). The terms peak I and peak II denote acylated, miceliar LTA and low-molecular-weight, apparently deacylated LTA, respectively (6, 12). The [3H]glycerol-labeled, micellar LTA used in the protoplast and whole cell incubation experiments was prepared (6, 12) from ca. 1.5 mg (dry weight) of cells grown in medium containing 20 iLCi of [2-3H]glycerol per ml and mixed with 4 mg of unlabeled LTA. By assuming that LTA constituted 1% of the dry weight of the labeled cells (7), we calculated t Present address: The Rockefeller University, New York, that the dry weight of the labeled LTA would be ca. NY 10021. 0.4% of the dry weight of the unlabeled LTA and thus 1176
DEACYLATION OF LIPOTEICHOIC ACID
VOL. 137, 1979
with 18 polyglycerolphosphate chains of LTAX. Thus, LTAX and, by inference, peak II LTA1 appeared to be deacylated; i.e., contain very few, if any, polyglycerolphosphate molecules with intact fatty acid(s). In similar experiments where some peak II (ca. 5% of total LTAJ) was found in aqueous phenol extracts of whole cells, the level of [1-'4C]acetate was at or below the background (data not shown). The extracellular material in the peak of very low electrophoretic mobility (peak "O", Fig. 1B) that contains [1'C]acetate but not [3H]glycerol has not yet been identified. It could represent extracellular lipid although it lacks glycerol, and excretion or turnover of lipid or free fatty acids was not noted in this organism in previous studies (6). Demonstration of an enzymatic activity for conversion of high-molecular-weight LTA to its low-molecular-weight form. To look for the presence of an appropriate enzymatic activity capable of converting acylated LTA to the apparently unacylated, low-molecular-weight form, radiolabeled acylated LTA was added to and incubated with growing cultures of intact streptococci and protoplasts. An LTA preparation was added to a final concentration of 100 ,ug/ml to cultures of intact streptococci, cultures of protoplasts, and to medium in which protoplasts had grown for 2 h. After incubation at 370C for 2 h, protoplasts and cells were removed by centrifugation. Samples (2.0 ml) of supernatant medium were dialyzed, lyophilized, and then resuspended in 100 ul of electrophoresis buffer. Ca. 95 and 93% of the added LTA was recovered in the supernatant from protoplasts and cells, respectively. Of this, in the case of LTA recovered from protoplast
its contribution to the total dry weight was considered to be negligible. The mixture was rechromatographed on the Sepharose 6B:Bio-Gel P-30 column system. A final specific activity of 6.25 x 105 dpm/mg of LTA was calculated. LTA with the electrophoretic mobility of peak II accounted for less than 0.1% of the total LTA as determined by agarose electrophoresis. This preparation was used to detect enzymatic activity capable of converting acylated LTA to its low-molecular-weight form. Radioactivity of samples was determined as described previously (6).
RESULTS Estimation of fatty acid content of LTA1 and LTAX. The result of agarose gel electrophoresis of an aqueous phenol extract of whole streptococci grown in the presence of [2-3H]glycerol and [1-'4Clacetate is shown in Fig. 1A. The ratio of 3H to C was 9:1 in peak I LTA. There was insufficient peak II to calculate a ratio for the radioactive material contained within this peak. Gel electrophoresis of concentrated, dialyzed spent culture medium showed a symmetrical [2-3H]glycerol profile at the position of peak II, as previously described for LTAX from this organism (6, 12). The gel slice representing the tip of the peak contained 6,400 dpm of 3H, but a mean of 30 dpm (0 to 10 dpm above the background) of 14C was counted in each slice under the 3H peak. If LTAx contained only a single fatty acid per molecule, in contrast to the four fatty acids covalently linked to each molecule of LTAi, then a minimum of 180 dpm of [114C]acetate would have been found in the same peak gel slice. (If more than one fatty acid remained attached to LTA., correspondingly greater counts would have been found.) This data has been interpreted as indicating that less than one fatty acid equivalent was associated -12
A.
*
0 12 B. 11 II .J* X0
010
2 0
4
6
~~~16
E-8
-
10 -8
-j 6
~~~~~~~~~~~E cl 122J 6_j
04 -I
8
DITAC FRO
0
1177
2
4
ORGI
6
8
LIL
4-
lam 0~~~0DITNEFO
10
DISTANCE FROM ORIGIN (cm)
0
2
4
RGN(m
6
8
10
DISTANCE FROM ORIGIN (cm)
FIG. 1. Agarose gel electrophoresis of LTAi (A) and LTAX (B) from a culture grown in the presence of [23H]glycerol and [1-'4C]acetate. Sample preparation and electrophoresis was carried out as previously described (6, 12). The two peak areas have been labeled I and II, and correspond to the positions of acylated micellar LTA and low-molecular-weight, apparently deacylated LTA, respectively (6, 12).
1178
KESSLER AND SHOCKMAN
J. BACTERIOL.
supernatants, ca. 15% was deacylated. The amounts of deacylated LTA in 20-p1 samples were calculated from the radioactivity contained in peak II (as defined in Fig. 1) after agarose electrophoresis, assuming an approximate mo0 E lecular weight of 10,000 for LTA. From electrophoresis of untreated samples, values of peak II 3 below 0.025 nmol/ml were calculated to be below the limits of detection and reported as not detectable. Very little deacylated LTA was de02 tected after 2 h of incubation with growing intact cells or with spent protoplast medium, but a substantial amount of deacylated LTA was de0 tected after 2 h of incubation with growing protoplasts (Table 1). The small amount of peak II found after incubation with spent medium may 90 60 120 30 0 have been due to contamination with a small TIME (minutes) number of protoplasts or a result of the small FIG. 2. Kinetics of conversion of exogenous LTA degree of lysis of the original protoplasts that Protoplasts of had been grown in this medium as previously to deacylated monomer by protoplasts. faecium were resuspended in protoplast medium reported for this system (10). The kinetics of S. 200 ug of [3H]LTA per ml. At the times conversion of exogenous LTA to the deacylated containing were removed and processed as samples indicated, monomer by growing protoplasts is shown in detailed in Materials and Methods. The amount of Fig. 2. LTA (200 ,ug/ml) was added to growing deacylated LTA is expressed as nmoles per 2 ml of protoplasts, and samples were removed at the the original culture. times shown and processed as described above. DISCUSSION Recovery of total LTA (acylated and deacylated) from the supernatants in these experiThe existence of an enzymatic activity for the conversion of micellar LTA (peak I) to unacyments was ca. 90%. lated LTA (peak II) has been demonstrated. By TABLE 1. Effect of incubation of LTA with growing exam ning the surrounding culture medium after incubation of peak I with growing protoplasts S. faecium cells, growing protoplats, and spent protoplast mediuma rather than extracting the protoplasts themselves, we were able to avoid the risk of chemical DeacylCulture turated hydrolysis that may accompany any extraction LTAC bidity Sample procedure. The enzyme appears to be cell bound (nmol/ (AOD)b because very little peak II could be detected ml) after incubation of peak I in spent protoplast 50 700 0.05 Cell supernatant medium. Contamination with a small number of 100 450 0.55 Protoplast supernatant or a small degree of lysis (10) may protoplasts 0.05 Spent protoplast medium account for the appearance of small amounts of NDd Uninoculated protoplast mepeak II in spent protoplast medium samples. dium Location of the enzyme on the outer surface of LTA was added to a final concentration of 100 pg/ the membrane is likely, considering the rhemml to growing intact cells, growing protoplasts, unin- brane location of LTAi (7, 15, 17). In the experoculated protoplast medium, and medium in which fatty acids of protoplasts had grown for 2 h but had been removed imental situation described, the by centrifugation. Each was incubated at 37°C for 2 h. the hydrophobic lipid moiety are presumed to Cells and protoplasts were separated from medium by be adsorbed to the exposed protoplast memcentrifugation (12,000 x g, 10 min, 4°C). The super- brane in a manner analagous to that of adsorpnatant media were dialyzed (2 ml per sample), lyoph- tion of LTA to erythrocytes (7, 9, 17). A memilized, and resuspended in 100 id of electrophoresis brane-bound enzyme then "deacylates" the buffer. A 20-pl amount of each was subjected to elec- LTA, releasing the very hydrophilic portion to trophoresis (see Fig. 2). the surrounding medium. The very small b AOD, Adjusted optical density. of peak II found after incubation of peak Calculation based on the dpm contained under amount in the I presence of growing cells may have peak II, a specific activity of 6.25 x 105 dpm/mg and resulted from restriction of access to the memmolecular weight of 10,000. d ND, Not detectable. Lower limit of detection was brane by the intact cell wall. In vivo, LTAi is a normal membrane component and thus, would 0.025 nmol/ml (see text). a
DEACYLATION OF LIPOTEICHOIC ACID
VOL. 137, 1979
be readily accessible to such an enzyme. The exact cleavage site(s) was (were) not determined in the present study. Thus, characterization of the enzymatic action as a true deacylation cannot be made. Cleavage at some other point(s) within the lipid moiety or between it and the polyglycerolphosphate chain may be responsible for the observed effects. Clarification of this point must await a complete structural analysis on the material found at the position of peak II after electrophoresis of LTA exposed to growing protoplasts. However, this material (i) elutes from Sepharose 6B columns at the volume expected for chemically deacylated LTA and for LTAX, (ii) contains glycerol and glycerol phosphate (5), and (iii) migrates to a position previously shown by agarose gel electrophoresis of labeled LTA and by crossed immuno- and affinoelectrophoresis to be characteristic of chemically deacylated LTA as well as native LTAX (6, 12). Furthermore, native LTAX was shown to contain little, if any, ['4C]acetate (Fig. 1B)-an indicator of the presence of fatty acids in micellar LTA;. Finally, all incorporated radioactive glycerol and 90% of incorporated radioactive acetate can be accounted for in lipid and LTA (6). Therefore, at the very least, enzymatic activity for conversion of acylated LTA to a molecule with electrophoretic properties of native LTA, from this organism has been demonstrated. The fact that only deacylated LTA has been found as LTAK, in contrast to that from other members of the Lactobacillaceae (5,8), suggests that two mechanisms, only one of them operating in S. faecium, may exist for the excretion of LTA. It may be that in a bacterium with very low or no loss of envelope components to the surrounding medium such as this particular strain (1, 6), LTA with intact fatty acids is not found extracellularly. Excretion preceded by deacylation may have a very different physiological function compared to loss of intact LTA. ACKNOWLEDGMENTS This research was supported by Public Health Service research grants Al 05044 from the National Institute of Allergy and Infectious Diseases and DE 03487 from the National Institute of Dental Research. We thank A. J. Wicken for many helpful suggestions during these studies.
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