Proc. Nat. Acad. Sci. USA Vol. 69, No. 1, pp. 233-237, January 1972

Teichoic Acid Hydrolase Activity in Soil Bacteria (Bacillus subtilis/sporulation/phosphodiesterase/polyamines/concanavalin A) EDMUND M. WISE, JR., RHONA S. GLICKMAN, AND ELLEN TEIMER Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111 Communicated by Jack L. Strominger, November 8, 1971

ABSTRACT Bacterial phosphodiesterases have been of the enzymic activity, especially under different conditions found that are capable of cleaving the backbone of teichoic of growth. acid. Such enzymes have not been reported previously. An aerobic, gram-negative, rod-shaped bacterium produc- MATERIALS AND METHODS ing this activity was detected and isolated by autoradi- ography of soil suspensions growing on minimal medium Bacterial Strains. B. subtilis ATCC 6051 (NCTC 3610, containing 32P-labeled Bacillus subtilis ATCC 6051 cell known as the Marburg strain and as the parent of 168 strains, walls as the sole phosphorus source. Broken-cell prepara- tions are capable of depolymerizing teichoic acids in media but not of W-23 strains) was obtained from the American of low ionic strength at near-neutral pH values. An active Type Culture Collection, Rockville, Md. The following teichoicase is also present in B. subtilis, ATCC 6051 (the Marburg derivatives were also used: strain 168I- was ob- Marburg strain), especially in sporulating cultures. tained from J. Spizizen; strain 168 BC8, a multiple auxotroph that produces a nonglucosylated, glycerol-containing teichoic Teichoic acids are phosphodiester-linked bacterial polymers acid in its cell wall, was obtained from F. E. Young (8), and of glycerol phosphate or ribitol phosphate, often with D- strain SB19 was obtained from H. V. Aposhian and M. alanine and sugar substituents as well. Glycerol teichoic acids Nishihara. B. subtilis strain W-23, containing a ribitol are found in association with the plasma membrane of most, teichoic acid in its cell wall, was obtained from H. V. Aposh- if not all, gram-positive bacteria, and glycerol or ribitol ian. Staphylococcus aureus H 52A2, a mutant that produces a teichoic acids also occur in the walls of many gram-positive ribitol wall teichoic acid without N-acetylglucosamine was bacteria. They may comprise as much of 10% of the dry obtained from A. N. Chatterjee (9). Strain TAE was isolated weight of bacterial cells. Since their discovery by Mitchell from soil obtained from Reading, Mass. and and Moyle (1) by Baddiley and coworkers (2), teichoic Preparation of Radioactive Wall and Radioactive Teichoic acids have been the subject of many structural, biosynthetic, and studies Little is known of Acid. Exponentially growing B. subtilis 6051 cells were labeled immunological (3, 4). their at with physiological role other than as bacteriophage attachment by growth 37°C in trypticase soy medium (13) in a sites (5-9), although roles in wall protection and in magnesium 32P, (2 mCi/liter). The cells were broken by sonication metabolism have been postulated (10). Since the diester Biosonik III apparatus, and the walls were separated by linkages of teichoic acids are resistant to all known enzymes differential centrifugation, extensively washed, boiled, treated structural with ribonuclease, deoxyribonuclease, and trypsin (Worthing- including nucleases, studies have depended on 2:1. blunt and laborious dissections by chemical methods. To ton) (14, 15), and extracted with chloroform-methanol meet the needs of structural studies and to find delicate Walls were then used as such, or teichoic acid was extracted methods to remove teichoic acid from membranes and from from the walls by shaking them with 10% trichloroacetic acid walls of living cells for physiological studies, we instituted a overnight at 4°C followed by precipitation with two volumes search for soil organisms that produce teichoicases. We of acetone at 0°C overnight. Radioactive walls of other or- report here the detection and isolation of a gram-negative ganisms were prepared in essentially the same way. Bio- bacterium, designated strain TAE, "teichoic-acid eater", synthetic '4C-labeled poly(glycerophosphate), and 'H-labeled that can incorporate phosphorus from agar containing a poly(ribitolphosphate), were gifts of D. R. D. Shaw. purified cell wall fraction of 32P-labeled Bacillus subtilis Isolation of Strain TAE. Washed soil suspension was spread ATCC 6051 (the Marburg strain) as sole phosphorus source. on Tris-low salts-glycerol minimal agar containing 32p_ This wall teichoic acid is poly(glycerophosphate), fully labeled walls of B. subtilis 6051 as the sole phosphorus source. substituted with a-glucoside residues on the 2 position of the Tris-low salts medium contains 10 mM NaCl, 5 mM KCl, glycerol (11, 12). This teichoicase is capable of depolymerizing 5 mM NH4Cl, 1 mM CaCl2, 1 mM MgCl2, 10 MM FeCl3, several teichoic acids. In addition, in the course of screening 2 mM Na2SO4, and 30 mM Tris (pH 7.4). 30 mM inorganic for teichoicase in other organisms, we have found considerable phosphate, 5 mM glucose,1 0 mM glycerol, or 1.5% agar were teichoicase activity in the organism that was the source of added as required. On each of several plates, 400,g (200,000 the walls used in the original screening, namely B. subtilis dpm) of radioactive walls were spread in a thin second layer 6051. That the enzymes in strain TAE and B. subtilis probably of agar. After incubation at 29°C for 4 days, Kodak No- have different physiological functions is apparent from the screen Medical x-ray film was placed on the agar plates with general nature of the organisms, i.e., not containing teichoic thin plastic Saran Wrap between the photographic film and acid versus containing teichoic acid, and from the magnitude the colonies. The film was exposed at room temperature for 233 Downloaded by guest on September 29, 2021 234 Biochemistry: Wise et al. Proc. Nat. Acad. Sci. USA 69 (1972)

an additional 1-3 days. The resulting autoradiographs 0.16 or 0.016 umol of 32P-labeled B. subtilis 6051 teichoic acid allowed detection and isolation of a bacterium that can use repeating units, 0.5 Mmol of MgCl2, and 0.5 Mmol of Tris- HC1 teichoic acid as its sole phosphorus source. Somewhat similar buffer, in a final volume of 50 ul. The final pH was 8.2. The colony autoradiographic methods have recently been used reaction mixture was incubated at 37°C for 30 min; it was by others to detect mutants (16, 17). then cooled in ice and aliquots were immediately spotted on strips of Whatman no. paper run Assay of Teichoicase in Strain TAE. Stationary cells of 41 and in solvent I (below). strain TAE grown in Tris-low salts, phosphate, glucose Chromatographic Systems. Aliquots of the incubation medium at 290C with shaking were sonicated, and the mixtures were spotted on strips of Whatman no. 41 paper. 40,000 X g (1 hr) supernatant was dialyzed against 1 mM Descending chromatograms were developed in one of several sodium maleate-1 mM EDTA (pH 6.5), and stored at 00C. solvent systems, in each of which teichoic acid remained at Aliquots were incubated with substrate at 290 C. The standard the origin. Solvent I was methanol-0.1 N formic acid, 7:3 assay consisted of 20-70 gg of supernatant protein, 0.16 (21). Chromatograms were run for 30-60 min; inorganic jsmol of phosphorus (microatoms of phosphorus) as B. s-ubtilis phosphate, a-glycerophosphate, and other teichoicase prod- 6051 teichoic acid, and 0.03 Armol of sodium maleate buffer, ucts had an Rf of 0.70. This solvent was used as the standard in a final volume of 30 MuI. The final pH was 6.5. The mixture assay system. Solvent II was 1-propanol-concentrated was incubated with shaking at 290C for 4 hr and the products ammonia-0.1 M EDTA 6:3:1. The running time was 15 hr. were determined as below. Organic and inorganic phosphorus In this solvent, a-glycerophosphate and the immediate were determined by the methods of Dryer et al. (18) or product of the B. subtilis teichoicase, denoted compound Ames (19), and protein is expressed as /g of protein equivalent X, ran 4.2 and 2.5 times as fast as phosphate, respectively. to bovine serum albumin by the method of Lowry et al. (20). Solvent III was tert-butanol-water-picric acid 80:20:4 (v/v/w) (22). Chromatograms were run for 15 hr and sepa- rated the following: inorganic phosphate (Rf 0.69), a-glycero- phosphate (Rf 0.65), and compound X (Rf 0.35). Solvent IV, methanol-concentrated ammonia- 0.03 MI EDTA 6:1:3 gives, in 2.5 hr, a good separation of inorganic phosphate

.~5*:'4i|| (Rf 0.40) from compound X (Rf 0.64) and a-glycerophosphate (Rf 0.68). Chromatograph strips v-ere scanned on a Nuclear Chicago Actigraph III apparatus. Other Materials. The plant lectin, concanavalin A, was obtained from CalBiochem. Yeast a-glucosidase was ob- tained from Sigma. p~~~~~~ RESULTS Isolation and properties of strain TAE Autoradiography showed that a number of the colonies growing on Tris-low salts-glycerol agar containing 32p_ 9~~~~~~~~~~~~~ labeled B. subtilis walls had accumulated an appreciable amount of radioactivity. Colonies of one organism continued to accumulate radioactivity when isolated. An autoradiograph of a streak plate of this teichoic acid hydrolyzing strain, denoted TAE, is shown in Fig. 1. Most of the colonies in the original plates inoculated with soil were merely taking up phosphorus compounds liberated from the teichoic acid by neighboring colonies. This was shown after isolation of Fiw 1. Autoradiograph of strain TAE on an agar streak plate. Colonies of strain TAE have hydrolyzed the 32P-labeled wall from several colonies. The rationale for using such a radioactive B. subtilis 6051 and incorporated the phosphorus. The crowded method for screening, after several others were tried, was colony area at the bottom shows less apparent radioactivity than the following. Attempts at enrichment with nonradioactive uncolonized areas, probably because of diffusion of phosphorus teichoic acid as the sole phosphorus source in liquid medium compounds down into the plate, with consequent absorption of were bound to fail, since teichoic acid would be degraded radiation. extracellularly to compounds that could benefit all the cells in a culture. With nonradioactive walls in agar to retard Assay of Teichoicase in B. subtilis 6051. Stationary cells of their diffusion, selection failed because the growth of or- B. subtilis 6051, grown in 1% tryptone (Difco), 0.5% NaCl ganisms was still slow compared with diffusion of phosphorus medium at 370C with shaking, were broken by sonication or compounds. In addition, even highly purified agars provided with glass beads in a Mickle apparatus. The broken-cell 1-5 ug of phosphorus per plate, as determined by chemical preparation was centrifuged at 40,000 X g for 20 mm, then assay. This amount of phosphate allowed for considerable at 100,000 X g for 1 hr, and the resulting supernatant fraction growth. was exhaustively dialyzed at 40C against 10 mM Tris- That strain TAE can stimulate the growth of neighboring 1 mM EDTA (pH 8.2), then against 10 mM Tris (pH 8.2). phosphorus-limited colonies is shown in Fig. 2A. Escherichia The crude enzyme preparation was normally stored at 00C coli was plated with strain TAE to give a satellite phenomenon and was stable for several weeks with little loss of activity. on phosphorus-limited agar to which purified B. subtilis walls The standard assay consisted of 30-100 ,ug of protein, either had been added. This method was initially tried as a selection Downloaded by guest on September 29, 2021 Proc. Nat. Acad. Sci. USA 69 (1972) Teichoicase Activity in Soil Bacteria 235

method for teichoicase producers, but failed. The extensive 0.5- background growth of very small colonies of E. coli seen in Fig. 2A at some distance from the TAE colonies was also seen on plates containing no wall or TAE or added inorganic E 0.4- phosphate. That phosphorus was indeed limiting was proven by the results with added phosphorus, as seen in Fig. 2B. F- That strain TAE can grow on wall phosphorus or inorganic cofactor Crude extracts of strain TAE degraded 82P-labeled teichoic requirement could be established. Ionic strengths less than acid of B. subtilis 6051 completely to inorganic phosphate, which was identified by chromatography in solvents 1, II, III, and IV. Teichoicase activity from B. subtilis 6051 When B. subtilis 6051 was examined for teichoicase, consider- able activity was found in extracts of cells grown in either rich broth or minimal media. Enzymic activity was highest in extracts of cells harvested in the stationary, sporulating phase. Dialyzed stationary-cell enzyme preparation, ob- tained from 100,000 X g supernatant fractions, gave maximal activity at pH 8.2 (data not shown). The cell extract also had a requirement for a divalent or polyvalent cation for activity. This requirement could be satisfied by Mg++, Ca++, putres- FIG. 2. Satellite colonies of E. coli about TAE colonies on cine, spermidine, or spermine, but not by Mn++ (Table 1). phosphorus-limited agar. 50 saline-washed TAE bacteria, grown While the true in vivo activator is unknown, it is interesting to in Tris-low salts, phosphate, glucose medium, plus 75 ,ug of phos- note that the concentration of polyamines reported for an phorus as B. subtilis 6051 walls (left) or plus 60 ,ug of phosphorus unnamed B. subtilis strain, containing mostly spermidine with as inorganic phosphate (right), were added to 5 ml of molten Tris- some putrescine, is about 8 mM (24). low salts, glucose agar and spread over 20 ml of hardened Tris- 10-fold lower concentrations of teichoic acid were used in low salts, glucose agar in a 150 X 15 mm Petri dish. After the plate was incubated 2 days at 29°C, 104 saline-washed E. coli, Table 1,B. Under these conditions, Ca++ was much less grown in Tris-low salts, phosphate, glucose medium, were added effective as a cofactor than Mg++. As also shown in Table 1, to 5 ml of molten Tris-low salts, glucose agar, spread over the the plant lectin, concanavalin A, which has an affinity for plates, and incubated 2 more days at 29°C. The colonies of strain a-glucosides (25), inhibits teichoicase activity. With 1 mg/ml TAE are about 2 mm in diameter. of concanavalin A added, which yielded a molar ratio of 1 Downloaded by guest on September 29, 2021 236 Biochemistry: Wise et al. Proc. Nat. Acad. Sci. USA 69 (1972)

concanavalin A molecule per 40 a-glucosylated teichoic acid repeating units (equivalent to about 2-4 teichoic acid polymer molecules), a precipitate was noted and the teichoicase activity was inhibited 47%. The B. subtilis teichoicase assay was proportional to protein concentration in the range 30-120 ,ug of protein and was linear with time. Preliminary studies have demonstrated 3 SC a Km 1 mM of units. ._S value below teichoic acid repeating E O.0 The chain length of the substrate is not known precisely, but 0 0 is thought to be 10-20 units long. Therefore, it is not possible 2a.E to give an alternative Km in terms of molarity of teichoic 4 6 acid molecules. w z EU. Only very low teichoicase activity is present in exponen- 4 a) tially growing cells (<30 nmol per mg of protein per hr). As 0 I-- shown in Fig. 4, teichoicase activity of the Marburg strain E 4 rises abruptly some 50-fold in early stationary phase, which 4 IC.)V strongly suggests a relationship of the enzyme to sporulation. 0 This relationship is further substantiated by the facts that w a. (a) media giving good sporulation have the highest teichoicase C,) activities (data not shown), and (b) certain early-sporulation mutants have very low teichoicase activities (data not shown). These high teichoicase values for B. subtilis during sporulation 4 8 12 16 1 24 are some 40-fold higher than the teichoicase specific activities HOURS in the gram-negative (nonsporulating) strain TAE when FIG. 4. B. subtilis teichoicase activity at different stages of both strains are assayed at comparable substrate concen- growth 500-ml shaken cultures of B. subtilis Marburg NCTC trations. 3610" were grown at 37°C in the sporulation medium of Schaeffer The radioactive products of freshly prepared, dialyzed at. (26), harvested at the times shown, broken by sonication, B. subtilis enzyme with its own 32P-labeled teichoic acid as and analyzed for teichoicase as in Table 1, B. This growth medium substrate are a mixture of inorganic phosphate and an gave the highest teichoicase activities of any used. Strain Mar- unidentified compound, X. Storage of the enzyme at 0°C for burg ATCC 6051 gave similar values. LI-W, specific activity; 00, A6oo. Arrow on abscissa indicates first observable re- fractility. TABLE 1. B. subtilis 6051 teichoicase 4 weeks results in enzymic activity that yields mostly com- Teichoicase activity, pound X at early time periods. Compound X labeled with Reaction mixture % of Complete 32p isolated from chromatograms developed in solvent II A. Complete 100 yields, quantitatively, 32pi as the sole labeled product with - Mg++ 4 Escherichia coli alkaline phosphatase, and only "2P-labeled + 10 mM Ca++ 94 glycerophosphates on heating with 1 N HCl at 100'C for +10mMMn++ <2 40 min. Compound X is not susceptible to yeast a-gluco- + 10 mM putrescine 61 sidase (27), presumably because of the phosphate group. + 10 mM spermidine 83 These preliminary results suggest that compound X is the + 10 mM spermine 115 monomer unit of this teichoic acid, namely a-glucosylglyc- Minus enzyme <2 erophosphate. We do not yet know the configuration of + enzyme boiled 1 min 45 the glycerophosphate moiety. + enzyme boiled 30 min <2 Substrains of B. subtilis Marburg also contained this % of Complete enzyme; these included 1681-, 168 BC8, and SB 19. The B. Complete 100 unrelated B. subtilis W-23, which has a ribitol teichoic -Mg++ + 10 mM Ca++ 24 acid in its cell wall, did not have significant teichoicase + concanavalin A. 0. 25 mg/ml 85 activity when assayed under these conditions with B. subtilis + concanavalin A. 1.0 mg/ml 47 6051 walls as substrate. + concanavalin A. 5.0 mg/ml <1 DISCUSSION A. The rate obtained with the complete mixture was 492 nmol The teichoicase activities of strain TAE and B. subtilis of product per mg protein per hr. The complete assay mixture Marburg have not been purified, so that we cannot say how consisted of B. subtilis 6051 extract containing 85,g of protein, many enzymes are involved in degradation, nor can we know 0.16,mol of 32P-labeled phosphorus (B. subtilis 6051 teichoic whether the activity seen is an accidental, but possibly useful, 1.0 Tris in a final acid), 0.5,umol MgCl2, and mol HCl buffer, of a for or is a volume of 50,u (pH 8.2). The mixture was incubated 30 mi at property nuclease, instance, caused by specific 370C and the products were chromatographed in solvent I. teichoicase. At least with the B. subtilis enzyme, high B. The rate obtained with the complete mixture was 484 nmol concentrations of DNA or RNA do not inhibit, which sug- of product per mg protein per hr. The mixture is similar to that gests a specificity for teichoic acid. In addition, when the in A, except that 0.016,mol of 32P-labeled teichoic acid and 29 Ag Marburg-strain enzyme is assayed with either B. subtilis of protein were used. W-23 teichoic acid (glucosylated ribitol phosphate polymer) Downloaded by guest on September 29, 2021 Proc. Nat. Acad. Sci. USA 69 (1972) Teichoicase Activity in Soil Bacteria 237

or one of several other teichoic acids (data not shown), the 7. Coyette, J. & Ghuysen, J. M. (1968) Biochemistry 7, 2385- activity is less than 2% of the activity obtained with its own 2389. 8. Young, F. E., Smith, C. & Reilly, B. E. (1969) J. Bacteriol. (B. subtilis Marburg) teichoic acid. Certainly teichoic acid 98, 1087-1097. must be produced in soil. Probably all gram-positive bac- 9. Chatterjee, A. N. (1969) J. Bacteriol. 98, 519-527. teria have at least a membrane teichoic acid, and many also 10. Heptinstall, S., Archibald, A. R. & Baddiley, J. (1970) have a teichoic acid in their walls so that, after such phosphate Nature 225, 519-521. 11. Glaser, L. & Burger, M. M. (1964) J. Biol. Chem. 239, 3187- compounds as nucleic acids and phytic acid, teichoic acids 3191. may represent a sizeable fraction of the bound organic phos- 12. Chin, T., Burger, M. M. & Glaser, L. (1966) Arch. Biochem. phorus produced in soil. The TAE enzyme, with its lack Biophys. 116, 358-367. of metal requirement and low production in rich medium, 13. Huff, E. & Silverman, C. S. (1968) J. Bacteriol. 95, 99-106. has properties suggestive of food gathering. The B. subtilis 14. Park, J. T. & Hancock, R. (1960) J. Gen. Microbiol. 224, 249-258. enzyme, while possibly having this function, may have 15. Wise, E. M., Jr. & Park, J. T. (1965) Proc. Nat. Acad. Sci. additional endogenous functions in wall teichoic acid turn- USA 54, 75-81. over (28, 29) or in possible teichoic acid turnover in cell 16. Zwaig, N. & Lin, E. C. C. (1966) Biochem. Biophys. Res. membranes. The high activity in sporulating cells strongly Commun. 22, 414-418. suggests a function in teichoic acid removal to produce 17. Martin, R. G. (1968) J. Mol. Biol. 31, 127-134. the 18. Dryer, R. L., Tammes, A. R. & Routh, J. I. (1957) J. Biol. changes seen in teichoic acid concentrations in some spores Chem. 225, 177-183. (30). It will be interesting to measure the production of the 19. Ames, B. N. (1966) in Methods in Enzymology, ed. Neufeld, B. subtilis enzyme under conditions when teichuronic acids E. F. & Ginsberg, V. (Academic Press, New York), Vol. are produced instead of teichoic acid (31), and in various VIII, p. 115. 20. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, classes of asporogenic stiains. R. J. (1951) J. Biol. Chem. 193, 265-275. This paper is dedicated to Eric G. Ball, a member of the 21. Shaw, D. R. D., Mirelman, D., Chatterjee, A. N. & Park, National Academy of Sciences, on the occasion of his retirement as J. T. (1970) J. Biol. Chem. 245, 5101-5106. Edward S. Wood Professor of Biological Chemistry, Harvard 22. Hanes, C. S. & Isherwood, F. A. (1949) Nature 164, 1107- Medical School. 1112. We thank Dr. D. R. D. Shaw for useful advice and Dr. M. 23. Baumann, P., Doudoroff, M. & Stanier, R. Y. (1968) J. Mandel for his determination of the guanine-plus-cytosine con- Bacteriol. 95, 1520-1541. tent of strain TAE. This work was supported by grant CA-08982 24. Herbst, E. J., Keister, D. L. & Weaver, R. H. (1958) Arch. from the National Cancer Institute and grant 71,908 from the Biochem. Biophys. 75, 178-185. American Heart Association. Preliminary reports appeared in 25. Goldstein, I. J. & So, L. S. (1965) Arch. Biochem. Biophy8. Bacteriol. Proc. (1970), p. 72 and Bacteriol. Proc. (1971), p. 49. 111, 407-414. 26. Schaeffer, P., Ionesco, H., Ryter, A. & Balassa, G. (1965) in 1. Mitchell, P. & Moyle, J. (1951) J. Gen. Microbiol. 5, 981- Mechanismes de regulation des activities cellulaires chez les 992. microorganisms (Centre Nat. Recher. Sci., Paris), p. 553. 2. Baddiley, J. (1959) Proc. Chem. Soc. London 1959, 177-182. 27. Halvorson, H. & Ellias, L. (1958) Biochim. Biophys. Acta 30, 3. McCarty, M. & Morse, J. I. (1964) Advan. Immunol. 4, 249- 28-40. 286. 28. Chaloupka, J., Rihova, L. & Kerkova, P. (1964) Folia 4. Archibald, A. R., Baddiley, J. & Blumson, N. L. (1968) in Microbiol. (Prague) 9, 9-15. Advances in Enzymology, ed. Nord, F. F. (Interscience, 29. Mauck, J., Chan, L. & Glaser, L. (1971) J. Biol. Chem. 246, New York), Vol. 10, p. 223. 1820-1827. 5. Glaser, L., Ionesco, H. & Schaeffer, P. (1966) Biochim. 30. Chin, T., Younger, J. & Glaser, L. (1968) J. Bacteriol. 95, Biophys. Acta 124, 415-417. 2044-2050. 6. Young, F. E. (1967) Proc. Nat. Acad. Sci. USA 58, 2377- 31. Tempest, D. W., Dicks, J. W. & Ellwood, D. C. (1968) 2384. Biochem. J. 106, 237-243. Downloaded by guest on September 29, 2021