Protoplasts of Streptococcus faecalis ATCC 9790 were produced with the aid of lysozyme, and the ability of these bodies to synthesize soluble, peptide cross-linked peptidoglycan (PG) fragments was examined. Lysozyme digests of PG isolated using gel filtration from the supernatant medium of protoplasts grown in the presence of ["C ]acetate and L-[8H Ilysine contained small amounts of PG having KD expected for peptide cross-linked dimers and trimers. Addition of benzyl penicillin (300 ,g/ml) to

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... owing protoplast cultures did not affect the net amount of PG fragments synthesized but resulted in inhibition of synthesis of dimer and trimer fractions by 27 and 59%, respectively. Failure of penicillin to completely inhibit the accumulation of the dimer fraction was attributed to the presence of atypical forms of dimer. In fact, the supernatant medium of penicillin-treated cultures did not contain detectable amounts of typical peptide cross-linked dimer. The degree of peptide cross-linkage of protoplast PG was at most only 13% of that found in walls isolated from intact streptococci. The relative amounts of monomers, dimers, and trimers synthesized during early and late stages of protoplast growth was approximately the same. Protoplasts synthesized soluble PG fragments in amounts which were of the same order of magnitude as that expected for insoluble PG produced by an equivalent amount of intact streptococci. Among the pivotal biochemical reactions in the synthesis of the rigid peptidoglycan (PG) layer in bacterial cell walls are the reactions which involve the addition of membrane-bound intermediates to established acceptor wall. These events occur outside the cellular permeability barrier. Consequently, effective integration with all the other events in the bacterial cell cycle is thought to require maintenance of a favorable spatial relationship between membrane-bound enzymes, substrates and intermediates, and the exocellular final acceptor (i.e., the wall; 6). Protoplasts of Streptococcus faecalis can be prepared with the aid of lysozyme, or by the action of native autolytic muramidase in the absence of added PG hydrolases, and grown in osmotically stabilized medium (9, 14). The preceding report (12) presents evidence which suggests that, as the amount of intact, acceptor wall remaining on streptococci decreases, the ability of these bodies to synthesize acid-'Present address: , Ind. 46202. precipitable (macromolecular) PG decreases to the point of being below detectable levels. Protoplasts prepared with the aid of lysozyme do, however, synthesize large quantities of soluble PG fragments which are released into the culture medium (12). After treatment of the supernatant medium with hen egg-white lysozyme, several molecular weight species of PG fragments were isolated using gel filtration. The majority of this material was recovered as disaccharide-peptide monomer, but approximately 20% of the soluble PG fragments was in the molecular weight range of bisdisaccharide-peptide dimers and trisdisaccharide-peptide trimers. The gel filtration profiles of the peaks corresponding to these substances were symmetrical and contained [V'C ]acetate: [8H ]lysine at a molar ratio of 2.0:1.0 throughout the peaks (12). Such a ratio was found in the cell wall PG of S. faecalis (P. Dezelee and G. D. Shockman, J. Biol. Chem., in press). Furthermore, the synthesis of materials present in both the dimer and trimer peaks was inhibited by cycloserine and vancomycin to the 410 on May 10, 2020 by guest http://jb.asm.org/ Downloaded from PEPTIDE CROSS-LINKS IN PG FRAGMENTS 411 same extent as disaccharide-peptide monomer (12) . The intent of this investigation was to characterize these presumed peptide crosslinked fragments and thereby to define more completely the ability of protoplasts to synthesize cross-linked PG fragments. MATERIALS AND METHODS Preparation of protoplasts of S. faecalis ATCC 9790 with the aid of lysozyme, growth of protoplasts in osmotically stabilizing medium containing L-[H Ilysine, L-["C ]lysine, and ["C lacetate (added individually or in combination), and isolation of soluble PG fragments from the supernatant medium of protoplast cultures or from cell walls of intact streptococci using gel filtration were performed as previously described (12). High-voltage paper electrophoresis was also carried out as described earlier (12) . Treatment of PG samples using Chalaropsis B muramidase (0.5 ug/ml) was performed in 0.05 M sodium acetate, pH 4.6, for 5 h at 37 C. ,B-Elimination. Desalted samples were treated with 2.0 ml of 0.05 N NaOH or 4 N NH4OH at 37 C for periods of time from 2 to 16 h. This treatment is known to catalyze the a-elimination of the lactyl-peptide moiety from N-acetylglucosaminyl-N-acetylmuramyl-peptide resulting in the liberation of the reduced disaccharide and, in this species, a mixture of N'-(a and ,B-aspartyl)lactyl-peptides (5, 18). Elimination of D-lactate or lactyl-peptides occurs only from free, reducing residues of muramic acid (5, 18). NaOH-treated samples, destined for electrophoresis, were desalted on a Sephadex G-10 column, whereas the NH4OH-treated preparations were desalted by evaporation in vacuo over H2S04 and P,O,. The disaccharide-peptide structural unit of the cell wall of S. faecalis consists of fl-1,4,N-acetylglucosaminyl-N-acetylmurarmyl-Na-(L-alanyl-D-isoglutaminyl)-N'-(D-isoasparaginyl)-L-lysyl-D-alanyl-(D-alanine) (5). The presence of the terminal D-alanine residue is variable and is dependent on the extent of the action of carboxypeptidase activity present in membranes of this species (4). Thus, the lactyl-peptide which will result from ,-elimination could be either a lactylpentaor a hexapeptide, and ,8-elimination of peptide cross-linked dimer could result in release of decaor undecapeptides. For the purpose of this investigation, the presence of either hexaand undecapeptide in protoplast monomer and dimer, respectively, is not pertinent. Thus, for the sake of simplicity, the peptide moieties of protoplast monomer and dimer will be referred to as pentapeptide and decapeptide, respectively. RESULTS Effect of penicillin on the synthesis of soluble PG fragments by protoplasts. A single preparation of protoplasts was inoculated into two flasks of osmotically stabilized medium. One flask contained ["C]lysine (0.83 MCi/ml) and 300 ,ug of penicillin G potassium per ml (Wyeth Laboratories, Radnor, Pa.), whereas the other flask contained onily L-['H ]lysine (2.5 ACi/ ml). In replicate experiments the procedure was the same except that the radioactive labels were reversed. After incubation until culture turbidities leveled off (about 140 min), the supernatants from control and penicillin-treated cultures were combined, digested with hen eggwhite lysozyme (E.C. 3.2.1.17; Boehringer-Mannheim), and subjected to gel filtration on connected columns of Sephadex G-50, G-50, and G-25. Quantitation of soluble PG fragments was calculated on the basis of the specific activity of ['H]-(control) and [14C]-(plus penicillin) lysine-labeled PG fragments and expressed as equivalents of PG disaccharidepentapeptide monomer. The presence of penicillin in the protoplast growth medium had no effect on either the rate or extent of turbidity increase of the cultures or on the total amount of soluble PG fragments synthesized (Table 1) . Also, the total amount of soluble PG fragments synthesized by protoplast cultureswas of the same order of magnitude as that expected for insoluble PG synthesized by an equivalent amount of whole cells (Table 1 ). In the presence of penicillin, the relative amounts of radioactive material in the dimer and trimer fractions were TABLE 1. Effect of penicillin (300 ug/ml) on PG synthesis by growing protoplastsa ntreated Penicillin treated Component PG % of PG % of % of (nequiv/ sm(nequiv/su cotl