Vol. 122, No. 1
JOURNAL OF BACTERIOLOGY, Apr. 1975, p. 152-158 Copyright © 1975, American Society for Microbiology
Printed in U.S.A.
Distribution of Teichoic Acid in the Cell Wall of Bacillus subtilis R. J. DOYLE,* M. L. McDANNEL, J. R. HELMAN, AND U. N. STREIPS Department of Microbiology and Immunology, University of Louisville Schools of Medicine and Dentistry, Louisville, Kentucky 40201
Received for publication 5 December 1974
Hydrolysis of the cell wall of Bacillus subtilis 168 by autolysins or lysozyme resulted in the exposure of glucosylated teichoic acid molecules as evidenced by increased precipitation of ["4C] concanavalin A. The number of concanavalin A-reactive sites increased significantly after only limited enzymatic digestion of the walls. Quantitative analyses of ["4C]concanavalin A-treated wall or wall hydrolysate complexes indicate that approximately one-half of the teichoic acid molecules are surface-exposed, whereas the remainder are probably embedded within the peptidoglycan matrix. Treatment of the cell walls with sodium dodecyl sulfate or Triton X-100 did not result in new concanavalin A-reactive sites. Partial autolysis diminished the ability of the cell walls to adsorb bacteriophage 025. Fluorescein-labeled concanavalin A bound intensely over the entire surface of growing B. subtilis 168 cells, suggesting that teichoic acid molecules are located on the total solvent-exposed surface area of the bacteria. The exact location and configuration of teichoic acids in the cell walls of gram-positive bacteria is still unknown. One important question that has remained unanswered is whether all of the cell wall teichoic acid molecules are surface exposed or whether some of the molecules are intercalated within the insoluble peptidoglycan network. Furthermore, with respect to bacilli, it is not known with certainty whether the teichoic acids are found in both hemispherical caps and rods. Several papers have appeared which deal peripherally with these problems. Burger (7) showed that by subjecting the cell walls of Bacillus subtilis 3610 and B. subtilis W-23 to lysozyme greater quantities of anti-teichoic acid antibody could be precipitated, suggesting that teichoic acids were arranged on both the surface and within the peptidoglycan matrix. Nermut (22) extracted the walls of B. megaterium M with hot formamide to remove the teichoic acid and found that the wall thickness decreased by approximately one-half. Nermut interpreted these results in terms of a two-layered cell wall structure, with the outer layer consisting of extractable teichoic acid and the inner layer consisting of nonextractable peptidoglycan. Birdsell et al. (Abstr. Annu. Meet. Am. Soc. Microbiol. 1972, G212, p. 65) found that thin sections of concanavalin A-treated B. subtilis 168 cell walls contained an outer fluffy layer and a smooth inner wall face. The fluffy, discontinu152
ous outer layer was absent when the walls were treated with the concanavalin A (Con A) inhibitor methyl-alpha-D-glucopyranoside or when walls containing non-glucosylated teichoic acid were employed. These findings suggested that in B. subtilis 168 at least a portion of the teichoic acid was located on the outer, but not the inner, surface of the cell wall. Recently, Weibull (26) showed that the cell wall of aldehyde-fixed, unstained B. subtilis ATCC 6051 possessed two electron-dense layers separated by an electron-transparent layer. One interpretation of these results is that the teichoic acid molecules are spatially restricted to the outer and inner cell wall surfaces. Earlier, Cole et al. (9), Granboulan and Leduc (17), and Glauert et al. (15) had presented evidence for a trilaminar wall structure in B. subtilis. These studies do not allow conclusions to be made regarding the exact location of teichoic acids. Recently, Millward and Reaveley (20) presented convincing electron microscope evidence for the occurrence of teichoic acid throughout the cell wall of B. licheniformis. Archibald et al. (4) proposed two models for the arrangement of the teichoic acid in the cell wall of Staphylococcus lactis. One model suggested that all of the teichoic acid was external to peptidoglycan strands, whereas the second model suggested a uniform distribution of teichoic acid throughout the wall. In the present report, we show that the enzymatic hydrolysis of B. subtilis cell walls
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results in the exposure of glucosylated teichoic acid molecules. Furthermore, by use of fluorescein-labeled Con A, it is shown that the teichoic acid is found on the entire surface area of the cell wall. MATERIALS AND METHODS Ceil cultures. B. subtilis 168 (obtained from F. E. Young) was grown in Spizizen minimal medium (24) according to previous reports (5, 10). Late-log-phase cells were harvested and used for the preparation of the cell walls. Col walls. Cell walls were prepared by disrupting the bacilli in a French press (American Instrument Company) at 18,000 lb/in' The walls were separated by differential centrifugation and washed 12 times with cold deionized water. Bacteriophage. A lysate of bacteriophage 0 (25) was prepared by the method of Birdsell and Doyle (5). Con A and derivatives. Con A was prepared according to Agrawal and Goldstein by using the Sephadex adsorption method (1). [14C]Con A was prepared by acetylating the protein with acetic [114Cjanhydride in half-saturated sodium acetate (2). Fluorescein-labeled Con A (FL-Con A) was prepared by adding 50 mg of fluorescein isothiocyanate (International Biological Supplies, Melbourne, Fla.) to 500 mg of Con A in 100 ml of 0.05 M phosphate buffer (pH 7.4) containing 0.1 M methyl-alpha-D-mannopyranoside. The solution was stirred at room temperature for 4 h. Any insoluble material was removed by centrifugation. The FL-Con A was precipitated by adding solid ammonium sulfate to 0.7 saturation. The precipitate was recovered by centrifugation, dissolved in water, and reprecipitated four times. The labeled Con A was then dialyzed against cold 1.0 M sodium chloride (eight changes of six liters) and finally against four changes of 0.05 M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride, pH 7.4. The product was then frozen in 1.0-ml portions until ready for use. The modification of Con A by either acetic anhydride or fluorescein isothiocyanate causes the protein to lose essential metals (2). Thus, just prior to use, the Con A derivatives were adjusted to 2 x 10-' M with respect to MnCl, and CaCl,. Interaction between FL-Con A and B. subtilis 168 cells. Mid-log-phase cells of B. subtilis 168 were harvested by centrifugation, washed three times with cold 0.04 M Tris-hydrochloride (pH 7.4), and suspended in the same buffer to a concentration of approximately 10' cells per ml. A 1-ml volume of cell suspension was mixed with 1 ml of FL-Con A (approximately 2.0 mg), incubated for 30 min at 4 C, and washed three times with Tris-hydrochloride. The pellet was suspended in several drops of buffer and 1 drop was placed on a cover slip. Excess liquid was withdrawn from the cover slip with a Pasteur pipette. The film was then air dried, fixed 10 min with acetone, and mounted with a solution containing 9 volumes glycerol and 1 volume of carbonate buffer, pH 9.0 (25). The slides were observed with a Zeiss standard RA microscope equipped with an Osram
153
mercury lamp, BG12 exciter filter, and barrier filter 53. Panatomic X film was employed and developed with Microdol X (Eastman Kodak Co., Rochester, N.Y.). Chemical analyses. Hexose was determined by the anthrone reagent of Mokrasch (21). Phosphorus was assayed by the method of Ames (3). Protein was estimated with the Folin reagent, described by Lowry et al. (19), with bovine plasma albumin as a standard. Scintillation counting. Samples for scintillation counting (0.5 ml) were added to 12 ml of Aquascint-IH (ICN, Chemical and Isotopes Div., Waltham, Mass.) in low-background glass vials and counted in a Packard Tri-Carb spectrometer. Standards of [I'CJCon A were employed to correct for quenching. Turbidity determinations. Turbidities of cell wall suspensions were obtained by use of a Spectronic 20 colorimeter employing 0.5-inch (ca. 1.27 cm) cylindrical glass cuvettes. Reagents. Lysozyme (crystallized three times; grade I) was purchased from Sigma Chemical Co., St. Louis, Mo. Sodium dodecyl sulfate (Sequanal grade) was purchased from Pierce Chemical Co., Rockford, ill. Triton X-100 and methyl-alpha-D-mannopyranoside were obtained from Sigma Chemical Co.
RESULTS Enzymatic release of teichoic acid. In previous studies (5, 10, 11) we have shown that Con A will interact with alpha-D-glucosylated teichoic acids. In the present study, use is made of Con A to probe the distribution of the teichoic acid in B. subtilis 168 cell walls. Cell walls were allowed to autolyze at pH 7.4 in 0.04 M Trishydrochloride buffer. At intervals, samples were withdrawn and mixed with [14C]Con A. If all of the teichoic acid molecules were exposed on the wall surfaces, it would be expected that a constant amount of [14C]Con A would precipitate as wall solubilization proceeds. On the other hand, if some of the teichoic acid molecules are buried within the peptidoglycan network and are released or exposed during autolysis, an increase in the amount of [14C]Con A precipitated would be expected. The latter is precisely what happens (Fig. 1). Before any autolysis occurred, approximately 0.49 mg of [14C ]Con A was precipitated by 0.5 mg of wall suspension. At 150 min, a time when autolysis is complete, the same weight of soluble autolysate precipitated approximately 0.87 mg of [14C]Con A. Thus, autolysis of wall solubilization results in exposure of Con A-reactive sites. The rate of increase in the amount of [14C]Con A precipitated does not appear to parallel the rate of autolysis. For example, at 15 min, autolysis was approximately 8% complete, as shown by the amount of soluble phosphorus, although a 21% increase in the amount of
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DOYLE ET AL.
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AUTOLYSIS TIME (MINUTES)
FIG. 1. Release of Con A-reactive teichoic acid from cell walls by autolysis. Autolysis was carried out at 23 C in 0.04 M Tris-hydrochloride buffer, pH 7.4. As autolysis proceeded, 1.0-ml samples were withdrawn, heat inactivated at 100 C for 15 min, and mixed with 1.0 ml of [14C]Con A (1,940 counts/min per mg). After an incubation period of 120 to 160 min, the complex was centrifuged and the amount of radioactivity remaining in the supernatant was determined by scintillation counting. The cell wall concentration was 0.5 mg/ml.
['4C]Con A precipitated was found. The relatively constant ratio of hexose/phosphate (Fig. 1) found in the supernatant of the autolysis medium indicates that a polysaccharide or nucleic acid contaminant was not preferentially solubilized from the walls. Similar results were obtained by digesting heat-inactivated cell walls with lysozyme (Fig. 2). In this experiment, it was found that 1.0 mg of cell walls would precipitate approximately 0.70 mg of ['4C]Con A. After complete hydrolysis, the digest precipitated approximately 1.41 mg of ['4C]Con A. Again, the rate of increased Con A precipitation is greater than the rate of hydrolysis. This is especially true at the early stages of digestion. At 10 min, hydrolysis was 15% complete, as measured by soluble hexose, but a 39% increase in the amount of Con A precipitated was observed. The ratio of soluble hexose/phosphorus was again essentially constant during lysozyme digestion of the walls. To show that the increased Con A binding to cell walls during autolysis or lysozyme digestion was due to an exposure of teichoic acid on the walls and not to the release of soluble teichoic acid, the following experiment was performed. Cell walls were allowed to autolyze at 37 C, samples were removed at intervals, and the remaining insoluble wall material was centrifuged and washed twice with water. This insured removal of soluble autolysis products. The partially autolyzed walls were then in-
teracted with ['4C]Con A (Table 1). Control wall samples (no autolysis) at a 1.0-mg concentration complexed with 0.66 mg of [14C]Con A. Under identical conditions, partially autolyzed walls bound higher quantities of Con A. For example, the wall material remaining after 60 min of autolysis complexed 1.08 mg of ['4C]Con A. Moreover, the increase in wall precipitability with Con A does not appear to correlate with a higher hexose content in the partially autolyzed walls (Table 1). When the walls were allowed to completely autolyze, the autolysate precipitated 1.22 mg of [14C]Con A, showing that 60 min of autolysis does not completely liberate all Con A-reactive sites. These data, along with the foregoing experiments, show that wall hydrol2~~~~~~~~~~~~~~~~~~~~~~~~C > * > *
-
*
~~~~~~~ ~~ >
9
~~0 8D
05r- 9( * 04
r
8(
E 3
i
71
0
60
Luu 02 e ° 61 co
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i
5(
tD
10
20
30
45
60
70
TIME (MIN)
FIG. 2. Effect of lysozyme on the interaction of cell walls with Con A. Procedural details are given in the legend to Fig. 1. The cell wall concentration was 1.0 mg/ml. The Con A concentration was 2.0 mg (specific activity of Con A was 6,228 counts/min per mg). TABLE 1. Binding of ['4C]Con A to partially autolyzed B. subtilis 168 cell walls" Autolysis (min)b
Con A bound (mg)
(Amol/mg)
0 5 15 40 60
0.66 0.86 0.89 0.94 1.08
0.96 1.01 1.00 1.01 1.09
Hexose
a Cell walls (1.0 mg) and [14C]Con A (2.0 mg) were mixed in 0.05 M Tris-hydrochloride buffer (pH 7.4) in a final volume of 2.0 ml. The mixtures were incubated 90 min and centrifuged, and the amount of radioactivity remaining in the supernatants was determined by scintillation counting (specific activity of ['4C]Con A was 2,436 counts/min per mg). bAutolysis (at 37 C) was carried out in 0.02 M (NH4)2CO3 (pH 8.6, T½/2 = 60 min). Samples were withdrawn at the indicated times, washed twice with cold deionized water, and lyophilized.
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CELL WALL TEICHOIC ACID
ysis is accompanied by an exposure of teichoic acid molecules. Interaction between Con A and modified B. subtilis cell walls. Most cell wall preparations contain autolysins (6) and nucleic acids (W. C. Brown and R. J. Doyle, unpublished data) which cannot be removed by washing with water. It is conceivable that such contaminants could mask teichoic acid molecules, resulting in a reduced interaction with Con A. Accordingly, cell walls were treated with agents that solubilize such potential contaminants, and then interacted with [14C]Con A (Table 2). Treatment of the walls with boiling 1% sodium dodecyl sulfate or cold 5 M LiCl did not result in a significant increase in Con A binding. In addition, when Triton X-100 was added to control walls followed by Con A, no new Con A-reactive sites were exposed. Treatment of the walls with agents that remove teichoic acids resulted in a marked decrease in the amount of Con A precipitated. For example, extraction of the walls with NaOH under nitrogen almost completely abolished the interaction with Con A. In addition, subjecting the walls to 5% trichloroacetic acid at 60 C for 90 min resulted in decreased Con A binding. Both the alkali and the 60 C trichloroacetic acid treatments removed large quantities of hexose from the walls. This experiment indicates that contaminants TABLE 2. Agglutination of modified B. subtilis 168 cell walls by Con A Cell walla 168 controld ................. 168 + 1% Triton X-100 ...... 168 extracted with sodium dodecyl sulfate ........... 168 extracted with 60C trichloroacetic acid ....... 168 extracted with alkali ..... 168 extracted with 5 M LiCl .
Con A Hexoseb H/Pc precipitated (mg) 0.96 0.96
0.81 0.81
0.49 0.50
1.10
0.85
0.50
0.19 0.09 1.00
0.89 1.1
0.21 0.08 0.52
0.86
[14C]Con A (1.0 mg; 4,100 counts/min) was mixed with 0.5 mg of cell wall suspension in 0.05 M Tris-hydrochloride (pH 7.4). Final volume was 2.0 ml. The suspension was incubated for 2 h at room temperature and centrifuged. The amount of Con A remaining in the supernatant was determined by scintillation counting. bHexose, as D-glucose equivalents, in the cell walls or extracted cell walls (micromoles per milligram). t H/P, Molar ratio of hexose to phosphorus in the cell walls. dThe cell walls contained 11% protein and 8.5% nucleic acid. After treatment with sodium dodecyl sulfate, the walls contained 1.9% protein and 1.9% nucleic acid. After extraction with LiCl, the walls contained 2.7% protein and 1.7% nucleic acid (W. C. Brown and R. J. Doyle, unpublished data). a
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do not mask Con A-reactive sites. However, removal of most of teichoic acid from the walls by alkali or trichloroacetic acid greatly reduces the extent of complex formation with Con A. Bacteriophage adsorption to partially autolyzed B. subtilis cell walls. The adsorption of bacteriophage 025 to B. subtilis depends on the presence of glucosylated teichoic acid in the cell wall (27). If autolysis exposes teichoic acid molecules on the wall surface, it would possibly result in more efficient adsorption of 025. B. subtilis 168 cell walls were autolyzed, and the insoluble residue was removed by centrifugation and washed twice with water. The partially autolyzed walls were then mixed with bacteriophage 025. The results show that phage adsorption did not increase, but was markedly lowered by autolysis (Table 3). After a 10-min autolysis period, phage binding was approximately onehalf that found for control walls. Continued autolysis decreased phage adsorption even further. Thus, although partial wall hydrolysis enhances Con A binding by exposing teichoic acids, phage adsorption is markedly diminished. Interaction between FL-Con A and B. subtilis 168 cells. Because Con A interacts specifically with the teichoic acid of B. subtilis 168, it should be possible to monitor the surface localization of the wall polymer by use of a fluoresTABLE 3. Effect of autolysis on bacteriophage binding sites in B. subtilis 168 cell wallsa Cell wall
(JLg)
Autolysis
Unadsorbed 425
time (min)
(PFU)Y x x x x
None 200 200 200
0 10 30
1.0 2.8 5.6 1.4
100 100 100
0 10 30
6.5 x 10' 1.0 x 103 2.0 x 103
40 40 40
0 10 30
1.5 x 103 2.6 x 103 4.4 x 101
106 102
102 103
a Cell walls were incubated with bacteriophage ,25, with gentle shaking, for 30 min at 37 C. Buffer for incubations was 0.05 M Tris-hydrochloride (pH 7.4). Total volumes were 1.0 ml. After incubation, the suspensions were centrifuged for 5 min at 25,000 x g. Supernatant samples were diluted and assayed for unadsorbed bacteriophage by the soft-agar overlay technique. Conditions for autolysis were 0.02 M (NH4) 2CO3 (pH 8.6) at 37 C. T,, was 47 min. b Plaque-forming units.
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DOYLE ET AL.
cent derivative of the protein. Con A, labeled with fluorescein isothiocyanate, was added to a washed suspension of mid-log-phase cells of B. subtilis. The results show that FL-Con A bound to the entire surface of the cells, although the septa appear to show an elevated uptake of the label (Fig. 3). Rods and hemispherical caps seem to bind FL-Con A equally well. Furthermore, discontinuities in FL-Con A binding to the cell surfaces are not apparent. Washing the cells with 0.05 M methyl-alpha-D-mannopyranoside, a potent Con A inhibitor (18), removes the FL-Con A. Moreover, cells such as B. subtilis gtaB290, which do not contain alphaD-glucosylated teichoic acids, could not be rendered fluorescent by FL-Con A.
DISCUSSION In this study, we have attempted to answer two fundamental problems relating to cell wall structure. First of all, what percentage of the wall teichoic acid is surface exposed and what percentage, if any, is located within the pepti-
FIG. 3. Interaction of FL-Con A with intact B. subtilis 168 cells. Exposure time was 4 min.
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doglycan matrix? Second, are there areas on the cell wall that do not contain teichoic acid? The use of the lectin Con A has enabled us to examine these questions. The results show that approximately one-half of the D-glucosylated teichoic acid molecules of B. subtilis 168 are surface exposed. Furthermore, teichoic acid appears to be located over the entire surface of vegetative cells. Solubilization of the B. subtilis 168 cell wall by either autolysins or lysozyme resulted in a significant increase in the amount of [14C]Con A precipitated (Fig. 1 and 2). Since twice as much Con A was precipitated after wall hydrolysis, it is suggested that one-half of the teichoic acid molecules are arranged within the peptidoglycan matrix. The data also suggest that the teichoic acid does not occur in tiers. If the teichoic acid were located in layers, it might be expected that the increase in Con A precipitation would occur as abrupt transitions. The data, however, show that the increased Con A precipitation occurred somewhat smoothly, without discontinuities. These results are in accord with the report of Millward and Reaveley (20), who extracted the teichoic acids from B. licheniformis and S. aureus and found that the typical trilaminar appearance of the cell walls was unchanged. In addition, our results are also consistent with the data of Burger (7), who suggested that cell wall teichoic acids of B. subtilis were located in a subsurface region. We found that on a dry weight basis partially autolyzed cell walls bound larger quantities of Con A than nonhydrolyzed walls (Table 1). We interpret these results in terms of Burger's hypothesis that limited hydrolysis of cell walls results in a general increased wall porosity. We have also noted that some variation in the amount of Con A precipitated occurs in different wall preparations. In preparations where care has been taken to prevent autolysis, smaller amounts of Con A are precipitated. Thus, to obtain reproducible results partial autolysis must be prevented. Scherrer and Gerhardt (23) have shown that monodisperse molecules larger than 1,200 daltons cannot penetrate the cell wall matrix of B. megaterium. Since Con A has a molecular weight of 110,000 at neutral pH, it is likely that the lectin is binding only those teichoic acid molecules that are completely exposed or external to the insoluble wall network. Extraction of the cell walls with sodium dodecyl sulfate or 5 M LiCl did not result in the exposure of new Con A-reactive sites. In addition, interacting the cell walls with Con A in the presence of 1% Triton X-100 did not alter binding (Table 2).
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Glew et al. (16) found that Triton X-100 exposes new Con A receptor sites on rat liver mitochondria. Thus, with respect to cell walls, all of the teichoic acid molecules that are on the wall surface are probably available for interaction with Con A. Treatment of the walls with agents that remove most of the teichoic acid significantly reduces binding with Con A (Table 2). This reduced Con A precipitation can be attributed to lower amounts of D-glucose in the extracted cell walls. Since wall hydrolysis results in the exposure of new Con A-reactive sites, it was thought that new bacteriophage receptor sites would be exposed as well. However, autolysis markedly reduced adsorption of 425, a bacteriophage that interacts specifically with glucosylated teichoic acids (Table 3). Earlier, Young (27) showed that autolysis reduced 425 adsorption to B. subtilis 168 cell walls. Our approach differed in one important detail. We used the insoluble wall remaining after partial autolysis, whereas Young used the total autolysate. Evidently, once the integrity of the wall has been challenged (autolysis), the phage receptor site geometry changes, resulting in decreased adsorption. This would suggest that bacteriophage 025 must recognize intact peptidoglycan as well as glucosylated teichoic acid for maximum adsorption. In this regard, Glaser et al. (14) found that the ribitol teichoic acid from B. subtilis W-23 would inactivate phage Mu providing the teichoic acid contained some contaminating peptidoglycan. The finding that FL-Con A stains intensely over the entire cell suggests that teichoic acids are distributed on all external regions of the cell wall (Fig. 3). Fan and co-workers (12, 13) have provided evidence suggesting that chemical differences exist between septa and sides of B. subtilis, although analytical data are lacking. We observed more intense staining by FL-Con A at septa but not at completely formed hemispherical caps. The apparent greater staining with FL-Con A at septal regions may be related to differences in teichoic acid content or may simply reflect the presence of a double wall. Cole has noted intense staining at division points for several rod-shaped bacteria by using fluorescein-conjugated antibodies (8). A reasonably clear picture has begun to emerge for the location of teichoic acid molecules in the cell wall of B. subtilis 168. Teichoic acid is located on the external, but not internal, face of the cell wall (Birdsell et al., Abstr. Annu. Meet. Am. Soc. Microbiol. 1972, G212, p. 65). Approximately one-half of the teichoic acid population is solvent exposed, distributed on all
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regions of the wall. The remainder of the teichoic acid population is inaccessible to large molecules, such as Con A, and is distributed throughout the peptidoglycan network. LITERATURE CITED 1. Agrawal, B. B. L., and I. J. Goldstein. 1967. Protein-carbohydrate interactions. VI. Isolation of concanavalin A by specific adsorption on cross-linked dextran gels. Biochim. Biophys. Acta 147:262-271. 2. Agrawal, B. B. L., I. J. Goldstein, G. S. Hassing, and L. L. So. 1968. Protein-carbohydrate interaction. XXfl. The preparation and properties of acetylated concanavalin A, the hemagglutinin of the jack bean. Biochemistry 7:4211-4218. 3. Ames, B. N., 1966. Assay of inorganic phosphate, total phosphate, and phosphatases, p. 115-118. In E. F. Neufeld and V. Ginsburg (ed.), Methods in enzymology, vol. 8, Academic Press Inc., New York. 4. Archibald, A. R., J. Baddiley, and J. E. Heckels. 1973. Molecular arrangement of teichoic acid in the cell wall of Staphylococcus lactis. Nature (London) New Biol. 241:29-31. 5. Birdsell, D. C., and R. J. Doyle. 1973. Modification of bacteriophage 025 adsorption to Bacillus subtilis by concanavalin A. J. Bacteriol. 113:198-202. 6. Brown, W. C. 1973. Rapid methods for extracting autolysins from Bacillus subtilis. Appl. Microbiol.
25:295-300. 7. Burger, M. M. 1966. Teichoic acids: antigenic determinants, chain separation, and their location in the cell wall. Proc. Nat. Acad. Sci. U.S.A. 56:910-917. 8. Cole, R. M. 1965. Symposium on the fine structure and replication of bacteria and their parts. m. Bacterial cell wall replication followed by immunofluorescence. Bacteriol. Rev. 29:326-344. 9. Cole, R. M., T. J. Popkin, R. J. Boylan, and N. H. Mendelson. 1970. Ultrastructure of a temperature-sensitive rod- mutant of Bacillus subtilis. J. Bacteriol. 103:793-810. 10. Doyle, R. J., and D. C. Birdsell. 1972. Interaction of concanavalin A with the cell wall of Bacillus subtilis. J. Bacteriol. 109:652-658. 11. Doyle, R. J., D. C. Birdsell, and F. E. Young. 1973. Isolation of the teichoic acid of Bacillus subtilis 168 by affinity chromatography. Prep. Biochem. 3:13-18. 12. Fan, D. P., and B. E. Beckman. 1973. Structural difference between walls from hemispherical caps and partial septa of Bacillus subtilis. J. Bacteriol. 114:790-797. 13. Fan, D. P., M. C. Pelvit, and W. P. Cunningham. 1972. Structural difference between walls from ends and sides of the rod-shaped bacterium Bacillus subtilis. J. Bacteriol. 109:1266-1272. 14. Glaser, L., H. lonesco, and P. Schaeffer. 1966. Teichoic acids as components of a specific phage receptor in Bacillus subtilis. Biochim. Biophys. Acta 124:415-417. 15. Glauert, A., E. Brieger, and J. Allen. 1961. The fine structure of vegetative cells of Bacillus subtilis. Exp. Cell Res. 22:73-85. 16. Glew, R. H., S. C. Kayman, and M. S. Kuhlenschmidt. 1973. Studies on the binding of concanavalin A to rat liver mitochondria. J. Biol. Chem. 248:3137-3145. 17. Granboulan, P., and E. Leduc. 1967. Ultrastructural cytochemistry of Bacillus subtilis. J. Ultrastruct. Res. 20:111-126. 18. Goldstein, I. J., C. E. Hollerman, and E. E. Smith. 1965. Protein carbohydrate interaction. II. Inhibition studies on the interaction of concanavalin A with polysaccharides. Biochemistry 4:876-883. 19. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R J. Randall. 1951. Protein measurement with Folin phenol
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reagent. J. Biol. Chem. 193:265-275. 20. Millward, G. R., and D. A. Reaveley. 1974. Electron microscope observations on the cell walls of some gram-positive bacteria. J. Ultrastruct. Res. 46:309-326. 21. Mokrasch, L. C. 1954. Analysis of hexose phosphates and sugar mixtures with the anthrone reagent. J. Biol. Chem. 208:55-59. 22. Nermut, M. V. 1967. The ultrastructure of the cell wall of Bacillus megaterium. J. Gen. Microbiol. 49:503-512. 23. Scherrer, R., and P. Gerhardt. 1971. Molecular sieving by the Bacillus megaterium cell wall and protoplast. J. Bacteriol. 107:718-735. 24. Spizizen, J. 1958. Transformation of biochemically defi-
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cient strains of Bacillus subtilis by deoxyribonucleate. Proc. Nat. Acad. Sci. U.S.A. 44:1072-1078. 25. Tkacz, J. S., C. B. Cybulska, and J. 0. Lampen. 1971. Specific staining of wall mannan in yeast cells with fluorescein-conjugated concanavalin A. J. Bacteriol. 105:1-5. 26. Weibull, C. 1973. Electron microscope studies on aldehyde-fixed, unstained microbial cells. J. Ultrastruct. Res. 43:150-159. 27. Young, F. E. 1967. Requirement of glucosylated teichoic acid for adsorption of phage in Bacillus subtilis 168. Proc. Nat. Acad. Sci. U.S.A. 58:2377-2384.
JOURNAL OF BACTERIOLOGY, Apr. 1975, p. 152-158 Copyright © 1975, American Society for Microbiology
Printed in U.S.A.
Distribution of Teichoic Acid in the Cell Wall of Bacillus subtilis R. J. DOYLE,* M. L. McDANNEL, J. R. HELMAN, AND U. N. STREIPS Department of Microbiology and Immunology, University of Louisville Schools of Medicine and Dentistry, Louisville, Kentucky 40201
Received for publication 5 December 1974
Hydrolysis of the cell wall of Bacillus subtilis 168 by autolysins or lysozyme resulted in the exposure of glucosylated teichoic acid molecules as evidenced by increased precipitation of ["4C] concanavalin A. The number of concanavalin A-reactive sites increased significantly after only limited enzymatic digestion of the walls. Quantitative analyses of ["4C]concanavalin A-treated wall or wall hydrolysate complexes indicate that approximately one-half of the teichoic acid molecules are surface-exposed, whereas the remainder are probably embedded within the peptidoglycan matrix. Treatment of the cell walls with sodium dodecyl sulfate or Triton X-100 did not result in new concanavalin A-reactive sites. Partial autolysis diminished the ability of the cell walls to adsorb bacteriophage 025. Fluorescein-labeled concanavalin A bound intensely over the entire surface of growing B. subtilis 168 cells, suggesting that teichoic acid molecules are located on the total solvent-exposed surface area of the bacteria. The exact location and configuration of teichoic acids in the cell walls of gram-positive bacteria is still unknown. One important question that has remained unanswered is whether all of the cell wall teichoic acid molecules are surface exposed or whether some of the molecules are intercalated within the insoluble peptidoglycan network. Furthermore, with respect to bacilli, it is not known with certainty whether the teichoic acids are found in both hemispherical caps and rods. Several papers have appeared which deal peripherally with these problems. Burger (7) showed that by subjecting the cell walls of Bacillus subtilis 3610 and B. subtilis W-23 to lysozyme greater quantities of anti-teichoic acid antibody could be precipitated, suggesting that teichoic acids were arranged on both the surface and within the peptidoglycan matrix. Nermut (22) extracted the walls of B. megaterium M with hot formamide to remove the teichoic acid and found that the wall thickness decreased by approximately one-half. Nermut interpreted these results in terms of a two-layered cell wall structure, with the outer layer consisting of extractable teichoic acid and the inner layer consisting of nonextractable peptidoglycan. Birdsell et al. (Abstr. Annu. Meet. Am. Soc. Microbiol. 1972, G212, p. 65) found that thin sections of concanavalin A-treated B. subtilis 168 cell walls contained an outer fluffy layer and a smooth inner wall face. The fluffy, discontinu152
ous outer layer was absent when the walls were treated with the concanavalin A (Con A) inhibitor methyl-alpha-D-glucopyranoside or when walls containing non-glucosylated teichoic acid were employed. These findings suggested that in B. subtilis 168 at least a portion of the teichoic acid was located on the outer, but not the inner, surface of the cell wall. Recently, Weibull (26) showed that the cell wall of aldehyde-fixed, unstained B. subtilis ATCC 6051 possessed two electron-dense layers separated by an electron-transparent layer. One interpretation of these results is that the teichoic acid molecules are spatially restricted to the outer and inner cell wall surfaces. Earlier, Cole et al. (9), Granboulan and Leduc (17), and Glauert et al. (15) had presented evidence for a trilaminar wall structure in B. subtilis. These studies do not allow conclusions to be made regarding the exact location of teichoic acids. Recently, Millward and Reaveley (20) presented convincing electron microscope evidence for the occurrence of teichoic acid throughout the cell wall of B. licheniformis. Archibald et al. (4) proposed two models for the arrangement of the teichoic acid in the cell wall of Staphylococcus lactis. One model suggested that all of the teichoic acid was external to peptidoglycan strands, whereas the second model suggested a uniform distribution of teichoic acid throughout the wall. In the present report, we show that the enzymatic hydrolysis of B. subtilis cell walls
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results in the exposure of glucosylated teichoic acid molecules. Furthermore, by use of fluorescein-labeled Con A, it is shown that the teichoic acid is found on the entire surface area of the cell wall. MATERIALS AND METHODS Ceil cultures. B. subtilis 168 (obtained from F. E. Young) was grown in Spizizen minimal medium (24) according to previous reports (5, 10). Late-log-phase cells were harvested and used for the preparation of the cell walls. Col walls. Cell walls were prepared by disrupting the bacilli in a French press (American Instrument Company) at 18,000 lb/in' The walls were separated by differential centrifugation and washed 12 times with cold deionized water. Bacteriophage. A lysate of bacteriophage 0 (25) was prepared by the method of Birdsell and Doyle (5). Con A and derivatives. Con A was prepared according to Agrawal and Goldstein by using the Sephadex adsorption method (1). [14C]Con A was prepared by acetylating the protein with acetic [114Cjanhydride in half-saturated sodium acetate (2). Fluorescein-labeled Con A (FL-Con A) was prepared by adding 50 mg of fluorescein isothiocyanate (International Biological Supplies, Melbourne, Fla.) to 500 mg of Con A in 100 ml of 0.05 M phosphate buffer (pH 7.4) containing 0.1 M methyl-alpha-D-mannopyranoside. The solution was stirred at room temperature for 4 h. Any insoluble material was removed by centrifugation. The FL-Con A was precipitated by adding solid ammonium sulfate to 0.7 saturation. The precipitate was recovered by centrifugation, dissolved in water, and reprecipitated four times. The labeled Con A was then dialyzed against cold 1.0 M sodium chloride (eight changes of six liters) and finally against four changes of 0.05 M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride, pH 7.4. The product was then frozen in 1.0-ml portions until ready for use. The modification of Con A by either acetic anhydride or fluorescein isothiocyanate causes the protein to lose essential metals (2). Thus, just prior to use, the Con A derivatives were adjusted to 2 x 10-' M with respect to MnCl, and CaCl,. Interaction between FL-Con A and B. subtilis 168 cells. Mid-log-phase cells of B. subtilis 168 were harvested by centrifugation, washed three times with cold 0.04 M Tris-hydrochloride (pH 7.4), and suspended in the same buffer to a concentration of approximately 10' cells per ml. A 1-ml volume of cell suspension was mixed with 1 ml of FL-Con A (approximately 2.0 mg), incubated for 30 min at 4 C, and washed three times with Tris-hydrochloride. The pellet was suspended in several drops of buffer and 1 drop was placed on a cover slip. Excess liquid was withdrawn from the cover slip with a Pasteur pipette. The film was then air dried, fixed 10 min with acetone, and mounted with a solution containing 9 volumes glycerol and 1 volume of carbonate buffer, pH 9.0 (25). The slides were observed with a Zeiss standard RA microscope equipped with an Osram
153
mercury lamp, BG12 exciter filter, and barrier filter 53. Panatomic X film was employed and developed with Microdol X (Eastman Kodak Co., Rochester, N.Y.). Chemical analyses. Hexose was determined by the anthrone reagent of Mokrasch (21). Phosphorus was assayed by the method of Ames (3). Protein was estimated with the Folin reagent, described by Lowry et al. (19), with bovine plasma albumin as a standard. Scintillation counting. Samples for scintillation counting (0.5 ml) were added to 12 ml of Aquascint-IH (ICN, Chemical and Isotopes Div., Waltham, Mass.) in low-background glass vials and counted in a Packard Tri-Carb spectrometer. Standards of [I'CJCon A were employed to correct for quenching. Turbidity determinations. Turbidities of cell wall suspensions were obtained by use of a Spectronic 20 colorimeter employing 0.5-inch (ca. 1.27 cm) cylindrical glass cuvettes. Reagents. Lysozyme (crystallized three times; grade I) was purchased from Sigma Chemical Co., St. Louis, Mo. Sodium dodecyl sulfate (Sequanal grade) was purchased from Pierce Chemical Co., Rockford, ill. Triton X-100 and methyl-alpha-D-mannopyranoside were obtained from Sigma Chemical Co.
RESULTS Enzymatic release of teichoic acid. In previous studies (5, 10, 11) we have shown that Con A will interact with alpha-D-glucosylated teichoic acids. In the present study, use is made of Con A to probe the distribution of the teichoic acid in B. subtilis 168 cell walls. Cell walls were allowed to autolyze at pH 7.4 in 0.04 M Trishydrochloride buffer. At intervals, samples were withdrawn and mixed with [14C]Con A. If all of the teichoic acid molecules were exposed on the wall surfaces, it would be expected that a constant amount of [14C]Con A would precipitate as wall solubilization proceeds. On the other hand, if some of the teichoic acid molecules are buried within the peptidoglycan network and are released or exposed during autolysis, an increase in the amount of [14C]Con A precipitated would be expected. The latter is precisely what happens (Fig. 1). Before any autolysis occurred, approximately 0.49 mg of [14C ]Con A was precipitated by 0.5 mg of wall suspension. At 150 min, a time when autolysis is complete, the same weight of soluble autolysate precipitated approximately 0.87 mg of [14C]Con A. Thus, autolysis of wall solubilization results in exposure of Con A-reactive sites. The rate of increase in the amount of [14C]Con A precipitated does not appear to parallel the rate of autolysis. For example, at 15 min, autolysis was approximately 8% complete, as shown by the amount of soluble phosphorus, although a 21% increase in the amount of
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DOYLE ET AL.
154
AUTOLYSIS TIME (MINUTES)
FIG. 1. Release of Con A-reactive teichoic acid from cell walls by autolysis. Autolysis was carried out at 23 C in 0.04 M Tris-hydrochloride buffer, pH 7.4. As autolysis proceeded, 1.0-ml samples were withdrawn, heat inactivated at 100 C for 15 min, and mixed with 1.0 ml of [14C]Con A (1,940 counts/min per mg). After an incubation period of 120 to 160 min, the complex was centrifuged and the amount of radioactivity remaining in the supernatant was determined by scintillation counting. The cell wall concentration was 0.5 mg/ml.
['4C]Con A precipitated was found. The relatively constant ratio of hexose/phosphate (Fig. 1) found in the supernatant of the autolysis medium indicates that a polysaccharide or nucleic acid contaminant was not preferentially solubilized from the walls. Similar results were obtained by digesting heat-inactivated cell walls with lysozyme (Fig. 2). In this experiment, it was found that 1.0 mg of cell walls would precipitate approximately 0.70 mg of ['4C]Con A. After complete hydrolysis, the digest precipitated approximately 1.41 mg of ['4C]Con A. Again, the rate of increased Con A precipitation is greater than the rate of hydrolysis. This is especially true at the early stages of digestion. At 10 min, hydrolysis was 15% complete, as measured by soluble hexose, but a 39% increase in the amount of Con A precipitated was observed. The ratio of soluble hexose/phosphorus was again essentially constant during lysozyme digestion of the walls. To show that the increased Con A binding to cell walls during autolysis or lysozyme digestion was due to an exposure of teichoic acid on the walls and not to the release of soluble teichoic acid, the following experiment was performed. Cell walls were allowed to autolyze at 37 C, samples were removed at intervals, and the remaining insoluble wall material was centrifuged and washed twice with water. This insured removal of soluble autolysis products. The partially autolyzed walls were then in-
teracted with ['4C]Con A (Table 1). Control wall samples (no autolysis) at a 1.0-mg concentration complexed with 0.66 mg of [14C]Con A. Under identical conditions, partially autolyzed walls bound higher quantities of Con A. For example, the wall material remaining after 60 min of autolysis complexed 1.08 mg of ['4C]Con A. Moreover, the increase in wall precipitability with Con A does not appear to correlate with a higher hexose content in the partially autolyzed walls (Table 1). When the walls were allowed to completely autolyze, the autolysate precipitated 1.22 mg of [14C]Con A, showing that 60 min of autolysis does not completely liberate all Con A-reactive sites. These data, along with the foregoing experiments, show that wall hydrol2~~~~~~~~~~~~~~~~~~~~~~~~C > * > *
-
*
~~~~~~~ ~~ >
9
~~0 8D
05r- 9( * 04
r
8(
E 3
i
71
0
60
Luu 02 e ° 61 co
°O
i
5(
tD
10
20
30
45
60
70
TIME (MIN)
FIG. 2. Effect of lysozyme on the interaction of cell walls with Con A. Procedural details are given in the legend to Fig. 1. The cell wall concentration was 1.0 mg/ml. The Con A concentration was 2.0 mg (specific activity of Con A was 6,228 counts/min per mg). TABLE 1. Binding of ['4C]Con A to partially autolyzed B. subtilis 168 cell walls" Autolysis (min)b
Con A bound (mg)
(Amol/mg)
0 5 15 40 60
0.66 0.86 0.89 0.94 1.08
0.96 1.01 1.00 1.01 1.09
Hexose
a Cell walls (1.0 mg) and [14C]Con A (2.0 mg) were mixed in 0.05 M Tris-hydrochloride buffer (pH 7.4) in a final volume of 2.0 ml. The mixtures were incubated 90 min and centrifuged, and the amount of radioactivity remaining in the supernatants was determined by scintillation counting (specific activity of ['4C]Con A was 2,436 counts/min per mg). bAutolysis (at 37 C) was carried out in 0.02 M (NH4)2CO3 (pH 8.6, T½/2 = 60 min). Samples were withdrawn at the indicated times, washed twice with cold deionized water, and lyophilized.
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CELL WALL TEICHOIC ACID
ysis is accompanied by an exposure of teichoic acid molecules. Interaction between Con A and modified B. subtilis cell walls. Most cell wall preparations contain autolysins (6) and nucleic acids (W. C. Brown and R. J. Doyle, unpublished data) which cannot be removed by washing with water. It is conceivable that such contaminants could mask teichoic acid molecules, resulting in a reduced interaction with Con A. Accordingly, cell walls were treated with agents that solubilize such potential contaminants, and then interacted with [14C]Con A (Table 2). Treatment of the walls with boiling 1% sodium dodecyl sulfate or cold 5 M LiCl did not result in a significant increase in Con A binding. In addition, when Triton X-100 was added to control walls followed by Con A, no new Con A-reactive sites were exposed. Treatment of the walls with agents that remove teichoic acids resulted in a marked decrease in the amount of Con A precipitated. For example, extraction of the walls with NaOH under nitrogen almost completely abolished the interaction with Con A. In addition, subjecting the walls to 5% trichloroacetic acid at 60 C for 90 min resulted in decreased Con A binding. Both the alkali and the 60 C trichloroacetic acid treatments removed large quantities of hexose from the walls. This experiment indicates that contaminants TABLE 2. Agglutination of modified B. subtilis 168 cell walls by Con A Cell walla 168 controld ................. 168 + 1% Triton X-100 ...... 168 extracted with sodium dodecyl sulfate ........... 168 extracted with 60C trichloroacetic acid ....... 168 extracted with alkali ..... 168 extracted with 5 M LiCl .
Con A Hexoseb H/Pc precipitated (mg) 0.96 0.96
0.81 0.81
0.49 0.50
1.10
0.85
0.50
0.19 0.09 1.00
0.89 1.1
0.21 0.08 0.52
0.86
[14C]Con A (1.0 mg; 4,100 counts/min) was mixed with 0.5 mg of cell wall suspension in 0.05 M Tris-hydrochloride (pH 7.4). Final volume was 2.0 ml. The suspension was incubated for 2 h at room temperature and centrifuged. The amount of Con A remaining in the supernatant was determined by scintillation counting. bHexose, as D-glucose equivalents, in the cell walls or extracted cell walls (micromoles per milligram). t H/P, Molar ratio of hexose to phosphorus in the cell walls. dThe cell walls contained 11% protein and 8.5% nucleic acid. After treatment with sodium dodecyl sulfate, the walls contained 1.9% protein and 1.9% nucleic acid. After extraction with LiCl, the walls contained 2.7% protein and 1.7% nucleic acid (W. C. Brown and R. J. Doyle, unpublished data). a
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do not mask Con A-reactive sites. However, removal of most of teichoic acid from the walls by alkali or trichloroacetic acid greatly reduces the extent of complex formation with Con A. Bacteriophage adsorption to partially autolyzed B. subtilis cell walls. The adsorption of bacteriophage 025 to B. subtilis depends on the presence of glucosylated teichoic acid in the cell wall (27). If autolysis exposes teichoic acid molecules on the wall surface, it would possibly result in more efficient adsorption of 025. B. subtilis 168 cell walls were autolyzed, and the insoluble residue was removed by centrifugation and washed twice with water. The partially autolyzed walls were then mixed with bacteriophage 025. The results show that phage adsorption did not increase, but was markedly lowered by autolysis (Table 3). After a 10-min autolysis period, phage binding was approximately onehalf that found for control walls. Continued autolysis decreased phage adsorption even further. Thus, although partial wall hydrolysis enhances Con A binding by exposing teichoic acids, phage adsorption is markedly diminished. Interaction between FL-Con A and B. subtilis 168 cells. Because Con A interacts specifically with the teichoic acid of B. subtilis 168, it should be possible to monitor the surface localization of the wall polymer by use of a fluoresTABLE 3. Effect of autolysis on bacteriophage binding sites in B. subtilis 168 cell wallsa Cell wall
(JLg)
Autolysis
Unadsorbed 425
time (min)
(PFU)Y x x x x
None 200 200 200
0 10 30
1.0 2.8 5.6 1.4
100 100 100
0 10 30
6.5 x 10' 1.0 x 103 2.0 x 103
40 40 40
0 10 30
1.5 x 103 2.6 x 103 4.4 x 101
106 102
102 103
a Cell walls were incubated with bacteriophage ,25, with gentle shaking, for 30 min at 37 C. Buffer for incubations was 0.05 M Tris-hydrochloride (pH 7.4). Total volumes were 1.0 ml. After incubation, the suspensions were centrifuged for 5 min at 25,000 x g. Supernatant samples were diluted and assayed for unadsorbed bacteriophage by the soft-agar overlay technique. Conditions for autolysis were 0.02 M (NH4) 2CO3 (pH 8.6) at 37 C. T,, was 47 min. b Plaque-forming units.
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DOYLE ET AL.
cent derivative of the protein. Con A, labeled with fluorescein isothiocyanate, was added to a washed suspension of mid-log-phase cells of B. subtilis. The results show that FL-Con A bound to the entire surface of the cells, although the septa appear to show an elevated uptake of the label (Fig. 3). Rods and hemispherical caps seem to bind FL-Con A equally well. Furthermore, discontinuities in FL-Con A binding to the cell surfaces are not apparent. Washing the cells with 0.05 M methyl-alpha-D-mannopyranoside, a potent Con A inhibitor (18), removes the FL-Con A. Moreover, cells such as B. subtilis gtaB290, which do not contain alphaD-glucosylated teichoic acids, could not be rendered fluorescent by FL-Con A.
DISCUSSION In this study, we have attempted to answer two fundamental problems relating to cell wall structure. First of all, what percentage of the wall teichoic acid is surface exposed and what percentage, if any, is located within the pepti-
FIG. 3. Interaction of FL-Con A with intact B. subtilis 168 cells. Exposure time was 4 min.
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doglycan matrix? Second, are there areas on the cell wall that do not contain teichoic acid? The use of the lectin Con A has enabled us to examine these questions. The results show that approximately one-half of the D-glucosylated teichoic acid molecules of B. subtilis 168 are surface exposed. Furthermore, teichoic acid appears to be located over the entire surface of vegetative cells. Solubilization of the B. subtilis 168 cell wall by either autolysins or lysozyme resulted in a significant increase in the amount of [14C]Con A precipitated (Fig. 1 and 2). Since twice as much Con A was precipitated after wall hydrolysis, it is suggested that one-half of the teichoic acid molecules are arranged within the peptidoglycan matrix. The data also suggest that the teichoic acid does not occur in tiers. If the teichoic acid were located in layers, it might be expected that the increase in Con A precipitation would occur as abrupt transitions. The data, however, show that the increased Con A precipitation occurred somewhat smoothly, without discontinuities. These results are in accord with the report of Millward and Reaveley (20), who extracted the teichoic acids from B. licheniformis and S. aureus and found that the typical trilaminar appearance of the cell walls was unchanged. In addition, our results are also consistent with the data of Burger (7), who suggested that cell wall teichoic acids of B. subtilis were located in a subsurface region. We found that on a dry weight basis partially autolyzed cell walls bound larger quantities of Con A than nonhydrolyzed walls (Table 1). We interpret these results in terms of Burger's hypothesis that limited hydrolysis of cell walls results in a general increased wall porosity. We have also noted that some variation in the amount of Con A precipitated occurs in different wall preparations. In preparations where care has been taken to prevent autolysis, smaller amounts of Con A are precipitated. Thus, to obtain reproducible results partial autolysis must be prevented. Scherrer and Gerhardt (23) have shown that monodisperse molecules larger than 1,200 daltons cannot penetrate the cell wall matrix of B. megaterium. Since Con A has a molecular weight of 110,000 at neutral pH, it is likely that the lectin is binding only those teichoic acid molecules that are completely exposed or external to the insoluble wall network. Extraction of the cell walls with sodium dodecyl sulfate or 5 M LiCl did not result in the exposure of new Con A-reactive sites. In addition, interacting the cell walls with Con A in the presence of 1% Triton X-100 did not alter binding (Table 2).
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Glew et al. (16) found that Triton X-100 exposes new Con A receptor sites on rat liver mitochondria. Thus, with respect to cell walls, all of the teichoic acid molecules that are on the wall surface are probably available for interaction with Con A. Treatment of the walls with agents that remove most of the teichoic acid significantly reduces binding with Con A (Table 2). This reduced Con A precipitation can be attributed to lower amounts of D-glucose in the extracted cell walls. Since wall hydrolysis results in the exposure of new Con A-reactive sites, it was thought that new bacteriophage receptor sites would be exposed as well. However, autolysis markedly reduced adsorption of 425, a bacteriophage that interacts specifically with glucosylated teichoic acids (Table 3). Earlier, Young (27) showed that autolysis reduced 425 adsorption to B. subtilis 168 cell walls. Our approach differed in one important detail. We used the insoluble wall remaining after partial autolysis, whereas Young used the total autolysate. Evidently, once the integrity of the wall has been challenged (autolysis), the phage receptor site geometry changes, resulting in decreased adsorption. This would suggest that bacteriophage 025 must recognize intact peptidoglycan as well as glucosylated teichoic acid for maximum adsorption. In this regard, Glaser et al. (14) found that the ribitol teichoic acid from B. subtilis W-23 would inactivate phage Mu providing the teichoic acid contained some contaminating peptidoglycan. The finding that FL-Con A stains intensely over the entire cell suggests that teichoic acids are distributed on all external regions of the cell wall (Fig. 3). Fan and co-workers (12, 13) have provided evidence suggesting that chemical differences exist between septa and sides of B. subtilis, although analytical data are lacking. We observed more intense staining by FL-Con A at septa but not at completely formed hemispherical caps. The apparent greater staining with FL-Con A at septal regions may be related to differences in teichoic acid content or may simply reflect the presence of a double wall. Cole has noted intense staining at division points for several rod-shaped bacteria by using fluorescein-conjugated antibodies (8). A reasonably clear picture has begun to emerge for the location of teichoic acid molecules in the cell wall of B. subtilis 168. Teichoic acid is located on the external, but not internal, face of the cell wall (Birdsell et al., Abstr. Annu. Meet. Am. Soc. Microbiol. 1972, G212, p. 65). Approximately one-half of the teichoic acid population is solvent exposed, distributed on all
157
regions of the wall. The remainder of the teichoic acid population is inaccessible to large molecules, such as Con A, and is distributed throughout the peptidoglycan network. LITERATURE CITED 1. Agrawal, B. B. L., and I. J. Goldstein. 1967. Protein-carbohydrate interactions. VI. Isolation of concanavalin A by specific adsorption on cross-linked dextran gels. Biochim. Biophys. Acta 147:262-271. 2. Agrawal, B. B. L., I. J. Goldstein, G. S. Hassing, and L. L. So. 1968. Protein-carbohydrate interaction. XXfl. The preparation and properties of acetylated concanavalin A, the hemagglutinin of the jack bean. Biochemistry 7:4211-4218. 3. Ames, B. N., 1966. Assay of inorganic phosphate, total phosphate, and phosphatases, p. 115-118. In E. F. Neufeld and V. Ginsburg (ed.), Methods in enzymology, vol. 8, Academic Press Inc., New York. 4. Archibald, A. R., J. Baddiley, and J. E. Heckels. 1973. Molecular arrangement of teichoic acid in the cell wall of Staphylococcus lactis. Nature (London) New Biol. 241:29-31. 5. Birdsell, D. C., and R. J. Doyle. 1973. Modification of bacteriophage 025 adsorption to Bacillus subtilis by concanavalin A. J. Bacteriol. 113:198-202. 6. Brown, W. C. 1973. Rapid methods for extracting autolysins from Bacillus subtilis. Appl. Microbiol.
25:295-300. 7. Burger, M. M. 1966. Teichoic acids: antigenic determinants, chain separation, and their location in the cell wall. Proc. Nat. Acad. Sci. U.S.A. 56:910-917. 8. Cole, R. M. 1965. Symposium on the fine structure and replication of bacteria and their parts. m. Bacterial cell wall replication followed by immunofluorescence. Bacteriol. Rev. 29:326-344. 9. Cole, R. M., T. J. Popkin, R. J. Boylan, and N. H. Mendelson. 1970. Ultrastructure of a temperature-sensitive rod- mutant of Bacillus subtilis. J. Bacteriol. 103:793-810. 10. Doyle, R. J., and D. C. Birdsell. 1972. Interaction of concanavalin A with the cell wall of Bacillus subtilis. J. Bacteriol. 109:652-658. 11. Doyle, R. J., D. C. Birdsell, and F. E. Young. 1973. Isolation of the teichoic acid of Bacillus subtilis 168 by affinity chromatography. Prep. Biochem. 3:13-18. 12. Fan, D. P., and B. E. Beckman. 1973. Structural difference between walls from hemispherical caps and partial septa of Bacillus subtilis. J. Bacteriol. 114:790-797. 13. Fan, D. P., M. C. Pelvit, and W. P. Cunningham. 1972. Structural difference between walls from ends and sides of the rod-shaped bacterium Bacillus subtilis. J. Bacteriol. 109:1266-1272. 14. Glaser, L., H. lonesco, and P. Schaeffer. 1966. Teichoic acids as components of a specific phage receptor in Bacillus subtilis. Biochim. Biophys. Acta 124:415-417. 15. Glauert, A., E. Brieger, and J. Allen. 1961. The fine structure of vegetative cells of Bacillus subtilis. Exp. Cell Res. 22:73-85. 16. Glew, R. H., S. C. Kayman, and M. S. Kuhlenschmidt. 1973. Studies on the binding of concanavalin A to rat liver mitochondria. J. Biol. Chem. 248:3137-3145. 17. Granboulan, P., and E. Leduc. 1967. Ultrastructural cytochemistry of Bacillus subtilis. J. Ultrastruct. Res. 20:111-126. 18. Goldstein, I. J., C. E. Hollerman, and E. E. Smith. 1965. Protein carbohydrate interaction. II. Inhibition studies on the interaction of concanavalin A with polysaccharides. Biochemistry 4:876-883. 19. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R J. Randall. 1951. Protein measurement with Folin phenol
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reagent. J. Biol. Chem. 193:265-275. 20. Millward, G. R., and D. A. Reaveley. 1974. Electron microscope observations on the cell walls of some gram-positive bacteria. J. Ultrastruct. Res. 46:309-326. 21. Mokrasch, L. C. 1954. Analysis of hexose phosphates and sugar mixtures with the anthrone reagent. J. Biol. Chem. 208:55-59. 22. Nermut, M. V. 1967. The ultrastructure of the cell wall of Bacillus megaterium. J. Gen. Microbiol. 49:503-512. 23. Scherrer, R., and P. Gerhardt. 1971. Molecular sieving by the Bacillus megaterium cell wall and protoplast. J. Bacteriol. 107:718-735. 24. Spizizen, J. 1958. Transformation of biochemically defi-
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cient strains of Bacillus subtilis by deoxyribonucleate. Proc. Nat. Acad. Sci. U.S.A. 44:1072-1078. 25. Tkacz, J. S., C. B. Cybulska, and J. 0. Lampen. 1971. Specific staining of wall mannan in yeast cells with fluorescein-conjugated concanavalin A. J. Bacteriol. 105:1-5. 26. Weibull, C. 1973. Electron microscope studies on aldehyde-fixed, unstained microbial cells. J. Ultrastruct. Res. 43:150-159. 27. Young, F. E. 1967. Requirement of glucosylated teichoic acid for adsorption of phage in Bacillus subtilis 168. Proc. Nat. Acad. Sci. U.S.A. 58:2377-2384.
