JOURNAL OF BACTERIOLOGY, Nov., 1965 Vol. 90, No. 5 Copyright © 1965 American Society for Microbiology Printed in U.S.A. Autolytic Mechanism for Spheroplast Formation in Bacillus cereus and Escherichia coli RAAM R. MOHAN, DONALD P. KRONISH, ROLAND S. PIANOTTI, RAY L. EPSTEIN,' AND BENJAMIN S. SCHWARTZ Department of Microbiology, Warner-Lambert Research Institute, Morris Plains, New Jersey Received for publication 11 June 1965 ABSTRACT MOHAN, RAAM R. (Warner-Lambert Research Institute, Morris Plains, N.J.), DONALD P. KRONISH, ROLAND S. PIANOTTI, RAY L. EPSTEIN, AND BENJAMIN S. SCHWARTZ. Autolytic mechanism for spheroplast formation in Bacillus cereus and Escherichia coli. J. Bacteriol. 90:1355-1364. 1965.-Spheroplasts of Bacillus cereus strain T and Escherichia coli B were prepared by incubating early log-phase cells in appropriate buffers and stabilizers for 3 hr at 30 and 37 C, respectively. Upon incuba- tion in 0.05 M tris(hydroxymethyl)aminomethane buffer osmotically stabilized with 16% polyethylene glycol at pH 7.5, 99% of the B. cereus cells formed spheroplasts; 90% of the E. coli cells were converted to spheroplasts in 0.4 M sodium acetate buffer osmotically stabilized with 1.6 M sucrose at pH 6.0. The extent of spheroplast formation was determined by phase-contrast microscopic examination, by measuring the rate of fall of optical density in the reaction mixture when subjected to osmotic shock, and by viable intact cell counts. The effect of a selected group of metabolic inhibitors on the autolytic system of B. cereus and E. coli has been examined. B. cereus and E. coli wall components comprising 26% of the dry weight of the original cellular material were recovered from dialyzed fractions by precipitation in 70% ethyl alcohol. Chemical and chromatographic analysis of cell-wall hydrolysates from B. cereus and E. coli indi- cated the presence of glucosamine, alanine, lysine, glycine, aspartic acid, diaminopi- melic acid, glutamic acid, and muramic acid. Spheroplast formation has been described in a cereus 130. The material, a protein, was precipi- wide variety of bacterial species (McQuillen, tated by (NH4)2SO4, was inactivated by heat or 1960), some fungi (Bachman and Bonner, 1959), trypsin treatment, and was irreversibly denatured and in at least one instance in higher plants at pH 4.0. In an osmotically stabilized system, (Cocking, 1960). Mitchell and Moyle (1957) showed that, under Methods for the physical, chemical, and enzy- certain conditions in the absence of lysozyme, matic preparation of spheroplasts from both suspensions of Staphylococcus aureus became gram-positive and gram-negative bacteria have osmotically sensitive, presumably by autodiges- been discussed in reviews by Salton (1961) and tion of the cell wall, but they did not examine the WVork (1961). Relatively little information, how- characteristics of the spheroplasts produced or the ever, is available on an autolytic mechanism of optimal conditions required for their production. spheroplast production. Nomura and Hosoda Kato et al. (1962) isolated a lytic factor desig- (1956) isolated an "autolysin" by (NH4)2SO4 nated Li, enzyme from culture supernatant liq- precipitation of autolysates of Bacillus subtilis uids of Flavobacterium sp., which exhibited lytic strain H, which caused lysis of B. subtilis and activity against intact cells and isolated cell walls B. megaterium but did not give rise to stable of S. aureus and Micrococcus lysodeikticus. spherophasts. Dark and Strange (1957) iso- The principal advantage of an autolytic system lated an enzyme from sporulating cells of for spheroplast production and recovery of de- B. cereus which was capable of rapidly and quan- graded cell wall lies in the elimination of any titatively converting cells of the same species to requirements for extraneous supplements, with spheroplasts. A lytic factor induced by ultra- the concomitant contamination of isolates. violet irradiation of B. cereus 569 was reported by The present report describes an autolytic sys- Csuzi and Kramer (1962) to induce lysis in B. tem operative in B. cereus strain T and Escherichia 1 Present address: Department of Bacteriology, coli B, optimal conditions for the production of University of Wisconsin, Madison. spheroplasts, and qualitative characterization of 1355 1356 MOHAN ET AL. J. BACTERIOL. recovered degraded cell-wall material. The pres- suspended in distilled water (100 ml), and disin- ent work was reported in preliminary form by tegrated at 10 C in a Raytheon 10-kc sonic oscilla- Kronish et al. p. 1960). tor for 15 min. The disintegrated cell mass was (Bacteriol. Proc., 63, centrifuged at 10,000 X g for 90 min, and the top white fluffy layer was carefully removed from the MATERIALS AND METHODS pellet. After three additional washings in distilled B. cereus T used in these studies was obtained water at 10,000 X g, the fluffy layer was treated with from H. 0. Halvorson, University of Wisconsin. deoxynuclease and ribonuclease at 40 C for 90 E. coli B was obtained from R. McDonald, Cornell min. The residual pellet was then washed eight University. times in distilled water. All operations except di- Growth of cells. B. cereus was grown in the salts- gestion were carried out at 4 C. The final washed glucose-yeast extract G medium of Stewart and pellet contained white fluffy cell-wall particulate Halvorson (1953). In later experiments, a water- material. In later experiments, equivalent yields dialyzed pancreatic digest of casein (Casitone, of cell wall were obtained by placing the cell mass Difco) replaced yeast extract. Cells were routinely in an Eppenbach colloid mill (model QV-6; grown overnight on a water-bath rotary shaker at Gifford-Wood Co., Hudson, N.Y.) together with 30 C, washed once in sterile distilled water, and 150 ml of distilled water and 90 ml of acid-washed inoculated in fresh medium to give an optical 120-, Superbrite glass beads (Minnesota Mining density of 0.05 at 660 mu (Bausch & Lomb Spec- and Manufacturing Co., St. Paul, Minn.). The tronic-20 colorimeter). colloid mill was run for 5 min at 17 C with a rotor- Cultures were incubated as above and grown to stator gap setting of 0.030 inches, according to the an optical density of 0.60 except where indicated. procedure of Garver and Epstein (1959). Subse- Cells were harvested by centrifugation at 5,000 X quent washing procedures and deoxynuclease and g and were washed once with distilled water. E. ribonuclease treatments were identical to those coli B was grown in the synthetic glucose-salts described above. Isolated cell walls prepared by medium of Davis and Mingioli (1950) at 37 C on a both methods were suspended in Tris or acetate rotary shaker (New Brunswick Scientific Co., New buffers and adjusted to an optical density of ap- Brunswick, N.J.) for 18 hr. A 3% inoculum was proximately 0.40 at 660 mp. Autolysis of the me- made to fresh medium and was incubated at 37 C chanically prepared cell walls from B. cereus and on a rotary shaker. Cells were harvested at an E. coli was determined by measuring the decrease optical density of 0.20 to 0 25 at 660 mu and washed in the optical density of the suspensions during once in 0.2 M NaHCO3. C ell pastes of both organ- incubation at 30 and 37 C, respectively. isms were examined for lytic activity and sphero- Isolation of enzymatically degraded cell wall from plast formation. the spheroplast-forming system. After incubation Lysis and spheroplast formation. B. cereus was of whole cells (until spheroplast formation was suspended in 0.05 M tris(hydroxymethyl)amino- complete), the spheroplast-forming system was methane (Tris) buffer (Sigma Chemical Co., St. centrifuged at 10,000 X g for 30 min to remove Louis, Mo.), pH 7.5, and was adjusted to an optical whole cells, undegraded cell wall, and debris. Cell- density of 0.70 at 660 m,p. Lysis of the cells was re- free supernatant fractions were made 70% with corded during incubation at 30 C by measuring fall respect to ethyl alcohol at -10 C. (Ethyl alcohol in optical density at 660 mA&. Spheroplasts were was cooled to -76 C before addition to prevent formed by suspending the cell paste in Tris buffer local heating and denaturation.) After standing as above, containing 16% (w/v) polyethylene overnight at 4 C, precipitates were centrifuged glycol 4000 (PEG; Union Carbide Chemicals Div., at 5,000 X g and washed twice with cold 70% ethyl New York, N.Y.). Lagerwerff, Ogata, and Eagle alcohol. The resultant pellet was water-soluble (1961) have discussed the osmotic properties of (with the exception of a very slight residue), and PEG. E. coli was suspended in 0.4 M sodium acetate was hydrolyzed in sealed ampoules in 4 N HCl at buffer at pH 6.0 and was incubated at 37 C. Lysis 121 C for 4 hr. Acid was removed by repeated was determined by measuring fall in optical evaporation to dryness prior to chromatography. density at 660 mp. The spheroplast-forming sys- Glucosamine was estimated according to the tem contained 1.6 M sucrose and 1.0 mg/ml of method of Rondle and Morgan (1955). Muramic MgSO4, in addition to sodium acetate as a sta- acid was separated from glucosamine by use of bilizing agent against osmotic lysis. 80% pyridine as the first solvent, followed by n- Mechanical preparation of cell walls. B. cereus butanol-acetic acid-water (68:15:30, v/v) in the and E. coli were grown in 10-liter batches in a New second dimension; its identity was established by Brunswick variable-drive fermentor (New Bruns- comparison with the RF value of an authentic wick Scientific Co.). Cultures were agitated by sample. Amino acid composition of hydrolysates horizontal impeller blades rotating at 250 rev/min was determined by two-dimensional paper chro- and were aerated at 0.2 liters per min through a disc-type sintered stainless-steel sparger.