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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.

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