Isolation and Characterization of Soluble Peptidoglycan from ...

Vol. 159, No. 2

Isolation and Characterization of Soluble Peptidoglycan from SeveralStrains of Streptococcus faeciumJOHN F. BARRETTt AND GERALD D. SHOCKMAN*

Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140

Received 20 March 1984/Accepted 20 May 1984

Two phenotypically autolysis-deficient strains of Streptococcusfaecium ATCC 9790 were shown to producehigh-molecular-weight, soluble, linear, uncross-linked peptidoglycan when incubated with benzylpenicillin in awall medium which permits cell wall synthesis (wall thickening) but not balanced growth. This high-molecular-weight s-peptidoglycan was shown to have a molecular weight of 46,000 to 54,000, lack peptide cross-links, andbe virtually devoid of accessory wall polymers. It was hydrolyzed by hen egg white lysozyme and theendogenous, autolytic N-acetylmuramidase of S. faecium, but was not attacked by proteinases. Chemicalanalyses of the polymer are consistent with the following structure, where n is the number of repeatingdisaccharide units:

(N-acetylglucosaminyl-j,1-4-N-acetylmuramic acid)n

L-alanine

D-glutamine

1e

L-lysine-D-isoasparagineD-alanine

D-alanine

Many bacterial species contain endogenous, autolyticenzymes (autolysins) which can hydrolyze bonds in theirown insoluble and protective cell wall peptidoglycan, result-ing in dissolution of the bacterial cell wall (6, 37). Theregulation of these potentially lethal enzymes has generatedconsiderable interest (6, 31, 37). Although the partial charac-terization of several autolytic enzyme systems has notyielded firm conclusions concerning their functional roles,such activities are thought to be involved in (i) cellulardivision (6, 18-21, 36, 49, 50); (ii) cell wall biosynthesis andremodeling of the cell wall (6, 11, 36); (iii) peptidoglycanturnover (3, 27, 38, 63); (iv) the final stages of physicalseparation of daughter cells (4, 10, 13, 14, 53, 57); (v) thelethal consequences of antibiotic inhibition of cell wallbiosynthesis (45, 50, 51, 58); (vi) genetic transformation (1,35, 44, 64); and (vii) flagellar morphogenesis (12).The autolytic enzyme system of Streptococcus faecium

ATCC 9790 (formerly S. faecalis ATCC 9790) appears to besimpler than the systems found in other bacteria, since asubstantial body of evidence indicates that only one bond inthe cell wall peptidoglycan, the ,-1,4 linkage between N-acetylmuramic acid and N-acetylglucosamine, is hydro-lyzed. This N-acetylmuramoylhydrolase (EC 3.2.1.17; SFmuramidase) activity (47, 52) is present in the cell wallfraction after cell disruption (34, 47, 52) and appears to besynthesized in a latent (zymogen) form, which is thentransported to appropriate sites on the cell wall, where it is

* Corresponding author.t Present address: Department of Biology, Washington Universi-

ty, St. Louis, MO 63130.

511

activated (47, 52). In vitro, a variety of proteinases canactivate the latent form of the autolysin (47, 52).

Until now, most assays for peptidoglycan hydrolases haveutilized: (i) intact cells, such as the cells of Micrococcusluteus (M. lysodeickticus) which are used for the convention-al assays for lysozyme activity; (ii) intact insoluble cell wallsor the insoluble peptidoglycan fraction of walls obtainedafter acid extraction of nonpeptidoglycan polymers; or (iii)low-molecular-weight substrates such as the products of henegg white lysozyme (HEWL) hydrolysis of bacterial walls(33) or oligosaccharides obtained from acid hydrolysates ofchitin (43, 62). Although insoluble cell and cell wall sub-strates have been extremely useful for many purposes, theyhave not permitted conventional kinetic analyses. Althoughthe products of HEWL hydrolysis of walls are useful forstudies of enzymes of other hydrolytic specificities, they arenot useful for studies of muramidases. Similarly, althoughchitin oligosaccharides have been extremely useful for de-tailed studies of the action of lysozymes, they are, in effect,analogs of the natural substrate(s) of these peptidoglycanhydrolases. Thus, for purposes of examining the action ofthe endogenous muramidase of S. faecium, we sought anatural soluble polymer that is, in large measure, the same asthat found in the cell wall of this organism. This polymer isthe soluble, linear, uncross-linked peptidoglycan (s-peptido-glycan) that is produced and secreted by penicillin-inhibitedcells of autolysis-defective strains of S. faecium. The s-peptidoglycan produced by S. faecium is similar to thatreported to be produced upon penicillin treatment of avariety of gram-positive bacteria, including M. luteus (29),Bacillus licheniformis (59), Brevibacterium divaricatum (25),

JOURNAL OF BACTERIOLOGY, Aug. 1984, p. 511-5190021-9193/84/080511-09$02.00/0Copyright © 1984, American Society for Microbiology

on April 2, 2018 by guest

http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

512 BARRETT AND SHOCKMAN

Bacillus subtilis (61), and Staphylococcus aureus (65). Wereport here the production, isolation, purification, and chem-ical characterization of the s-peptidoglycan produced byseveral strains of S.faecium. In the accompanying paper (2),these polymers are used as substrates for kinetic and otheranalyses of the mechanism of action of the SF muramidase.

MATERIALS AND METHODS

Bacterial strains and growth measurements. Lyophilizedcells of the parental strain of S. faecium and two mutantstrains of S. faecium, Lyt-14 and Aut-3, with autolysis-defective phenotypes (4, 53) were suspended in complexmedium (S broth) consisting of (per liter of water) 10 g oftryptone (Difco Laboratories, Detroit, Mich.), 10 g of yeastextract (Difco), 20 g of glucose, 0.41 g of KH2PO4, 0.35 g ofK2HPO4, and 0.3 M sodium phosphate, pH 6.8, at 37°C.Cultures were then incubated overnight and plated on solidmedium consisting of S broth with 1.4% agar. For routineuse, inocula were prepared from isolated colonies grown onsolid medium. Both mutant strains of S. faecium weremonitored for reversion by periodically plating the strains onsolid medium containing S broth and 1.4% agar, with anoverlay of S broth containing ethylene oxide-killed cells (1mg/ml) of either M. luteus NCTC 2665 or S. faecium in 1.4%agar (4). Mutant phenotypes were identified by their failureto produce a clear halo around colonies due to dissolution ofbacteria in the agar overlay. Only cultures growing atdoubling times of 30 to 33, 33 to 35, and 65 to 70 min in Sbroth for parental, strain Lyt-14, and strain Aut-3 cultures,respectively, were used to produce s-peptidoglycan or toprovide cell walls for extraction of the SF muramidase.Growth of cultures was monitored turbidimetrically with aColeman model 14 spectrophotometer (Coleman InstrumentCo., Maywood, Ill.) at 675 nm, and the absorbance at 675 nmwas adjusted to agree with Beer's law (56). One AdjustedOptical Density (adjusted absorbance x 103) unit is equiva-lent to 0.39 jig of cellular dry weight per ml of culture.

Gel filtration and paper chromatography. Gel filtrationswere carried out on upward-flow columns of Sephadex G-100 (37 by 2.5 cm, V, = 182 ml; Pharmacia Fine Chemicals,Inc., Piscataway, N.J.), Sephadex G-200 (31 by 2.5 cm, V, =152 ml), Ultrogel AcA 34 (100 by 2.5 cm, V, = 491 ml; LKB,Gaithersburg, Md.), and Bio-Gel P-2 (38 by 1.25 cm, V, = 47ml; Bio-Rad Laboratories, Richmond, Calif.) calibrated withblue dextran (Pharmacia) and glucose. Paper chromatogramswere run at room temperature in solvent 1 (butanol-aceticacid-water [4:1:5, vol/vol/vol, upper phase]) or in solvent 2(butanol-acetic acid-water [3:1:1, vol/vol/vol]). All chro-matograms were run on Whatman 1 paper.

Incorporation of radiolabeled precursors into whole cells.Incorporation of 14C- or 3H-labeled precursors into trichloro-acetic acid-insoluble macromolecules was measured as de-scribed previously (39). Precipitates on filters were dissolvedin 0.5 ml of 90% NCS (Amersham Corp., Arlington Heights,Ill.) in low-background glass scintillation vials (BeckmanInstruments, Inc., Fullerton, Calif.) at 50°C for 2 h andcounted as described below.

Scintillation counting. Aqueous samples (taken from gelfiltrations or eluted from paper chromatograms) were count-ed in Formula 947 (New England Nuclear Corp., Boston,Mass.). Precipitates on glass fiber filters were dissolved in90% NCS and counted in 5 ml of a toulene-based scintillationcocktail (Amersham). All samples were counted with an

Intertechnique model SL3000 scintillation counter (IN/USCo., Fairfield, N.J.) with efficiencies of 40 to 50% for 14C

and 25 to 30% for 3H. Corrections for overlap were madewith an external standard and external channel ratios.

Chemicals and reagents. Benzylpenicillin (Pen G) was agenerous gift of Wyeth Laboratories, Radnor, Pa. Bovineplasma albumin was purchased from Armour Pharmaceuti-cal Co., Kankakee, Ill.; subtilisin BPN' (Nagarse, EC3.4.4.16) was from Nagase & Co., Ltd., Osaka, Japan;chymotrypsin (EC 3.4.4.5) was from Worthington Diagnos-tics, Freehold, N.J.; trypsin (EC 3.4.4.4; 2x crystallized)was from GIBCO Laboratories, Grand Island, N.Y.; andHEWL (22,000 U/mg; EC 3.2.1.17) was from Boehringer-Mannheim (GmbH, Federal Republic of Germany) D-[6-3H]Glucose (24 mCi/mmol), D-[U-14C]glucose (336 mCi/mmol), L-[U-14C]lysine (354 mCi/mmol), and DL-[1-14C]ala-nine (305 mCi/mmol) were from Amersham. L-[4,5-3H]lysine(64.5 Ci/mmol) and [3H]NaBH4 (247.8 Ci/mmol) were fromNew England Nuclear Corp. All other chemicals were ofreagent grade. Distilled water was used for all dialyses, gelfiltrations, and assays.

Production of s-peptidoglycan. The medium used for pro-duction of s-peptidoglycan from S. faecium was slightlymodified from the wall mediurp of Shockman et al. (48) andconsisted of glucose, 5.6 mM; sodium acetate, 14.5 mM;(NH4)2504, 7.8 mM; MgCl2, 1.0 mM; MnCl2, 1.0 mM; NaCl,0.17 mM; FeSO4, 0.36 ,uM; potassium phosphate, 80 mM; L-glutamic acid, 2.0 mM; L-aspartic acid, 0.75 mM; L-lysine,0.68 mM; DL-alanine, 0.75 mM; L-cysteine, 1.7 mM; uracil,0.27 mM; guanine, 0.2 mM; and thiamine, 3.0 ,uM. The pHwas adjusted to 6.5 with 0.1 N HCI. In some experiments anoriginal component of the medium (48), 0.3 M sodiumphosphate, was added. This medium was sterilized by filtra-tion through a 0.45-,um membrane filter (Millipore Corp.,Bedford, Mass.) and stored for no longer than 2 weeks at40C.

After growth of 1-liter cultures in S broth to mid-exponen-tial phase (0.16 to 0.2 mg of cellular dry weight per ml) at370C, cells were harvested (20,000 x g 10 min, 32°C),washed by suspending the cells in 250 ml of prewarmed(32°C) 0.08 M sodium phosphate (pH 6.8), centrifuged(20,000 x g 10 min, 320C), and suspended in 200 ml ofprewarmed (37°C) wall medium. After 5 min at 37°C, Pen G(usually 50 ,ug/ml) and, when appropriate, radiolabeled pre-cursors were added. In the experiments in which a radiola-beled compound (e.g., [3H]glucose, [14C]glucose, [3H]lysine,[14C]lysine, or [14C]alanine) was added, the concentrationsof the corresponding unlabeled medium components weresometimes reduced (as indicated in figure legends) to facili-tate more efficient incorporation of the radiolabeled com-pound into the s-peptidoglycan. Routinely, 1.25 x 1012 cellsper ml were incubated in the wall medium for 1 h at 37°C.The cells were removed by centrifugation (20,000 x g, 10min, at 4°C) and washed once in 100 ml of 0.08 M sodiumphosphate, pH 6.8 (chilled to 4°C). Culture supernatants andwashes were pooled, heated to 95 to 100°C for 7 min toinactivate endogenous muramidase activity, and filtered(0.45-,um pore diameter) to remove particulate matter anddenatured protein. The supernatant was then dialyzed (fourtimes against 8 liters of water, with 5,000-molecular-weightretention dialysis tubing, at 4°C), and the retentate was

lyophilized. This dried material was dissolved in 3 ml ofwater, filtered (0.22-,um filter), and then filtered on Sepha-dex G-100. The column was eluted with water (20 ml/h), andfractions containing s-peptidoglycan (detected by radioactiv-ity or absorption at 225 nm) were pooled and lyophilized.SF muramidase. The SF muramidase from each of the

strains (wild type, Lyt-14, and Aut-3) was extracted and

J. BACTERIOL.


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

SOLUBLE PEPTIDOGLYCAN OF S. FAECIUM 513

affinity purified by adsorption and elution from concanavalinA-Sepharose as described previously (24).Chemical and physical analyses. Amino acid content was

determined after hydrolysis in 6 N HCl for 18 h at 100°C witha Beckman model 119 automatic amino acid analyzer. Totalamino sugar content was determined by a modified Morgan-Elson reaction (17) after hydrolysis in 3 N HCI for 4 h at95°C. The content of phosphorus (26), methylpentose (7),and neutral sugars (8) was determined.The molecular size of the native s-peptidoglycan or lower-

molecular-weight derivatives was determined by elutionprofiles over columns of AcA 34, Sephadex G-200, orSephadex G-100, each calibrated with dextran T-70 (0.04%,Pharmacia). The number average of glycan chain lengths ofpolymers was determined both by the Morgan-Elson reac-tion for amino sugars (17) and by reducing groups by asemimicro modification of the Park and Johnson method (32,55) before and after acid hydrolysis (3 N HCl, 4 h, 95°C) andbefore and after complete enzymatic hydrolysis of the s-peptidoglycan by the SF muramidase.Sodium borotritide labeling of the reducing terminus.

[3H]NaBH4 was used to selectively label the reducing termi-ni of s-peptidoglycans and their derivatives (28). Unlabeledhigh-molecular-weight s-peptidoglycan (0.16 mg, ca. 4.5nmol of chains), produced by the Lyt-14 strain, was incubat-ed with activated (0.2 ,ug of trypsin per ml) SF muramidase(85 U/ml) in 10 mM sodium phosphate, pH 6.8, at 37°C for 18h. This hydrolysate was applied to a Bio-Gel P-2 column,and the peak fractions (detected by absorption at 225 nm)were pooled and lyophilized. Equal amounts of the s-peptidoglycan and the hydrolysate were reduced, with 50,uCi of [3H]NaBH4 added to each sample in 200 ,ul of 10 mMNaOH in a closed vial. After 8 h at 4°C, the reaction wasstopped by the addition of 80 mM acetone (final concentra-tion). The reduced products were separated by paper chro-matography in solvent 1 (descending, 48 h), eluted withwater, lyophilized, and hydrolyzed with 300 p.l of 6 N HClfor 12 h at 100°C in a sealed glass ampoule. The hydrolysateswere dried over NaOH, and HCl was removed by dissolvingthe residues in 0.5 ml of water and drying them over NaOHthree times. Finally, portions of the acid hydrolysates wereseparated by paper chromatography in solvent 2 (ascending,4 h). Products were identified with [3H]muramicitol and[3H]glucosaminitol as standards, prepared from N-acetyl-muramic acid and N-acetylglucosamine by reduction with[3H]NaBH4 (28).

RESULTSPresumptive evidence for production of s-peptidoglycan by

autolysis-defective strains of S. faecium. Previous data indi-cated that S. faecium has an active autolytic muramidase(47, 52). Therefore, two autolysis-defective strains (Lyt-14and Aut-3) were used to produce s-peptidoglycan in wallmedium containing Pen G. The initial experiments weremodeled after work reported with other gram-positive organ-isms in which Pen G inhibited the transpeptidase reaction,thereby allowing secretion of s-peptidoglycans (25, 29, 59).

Incubation of the Lyt-14 or Aut-3 strain for 1 h in wallmedium containing 10 jig of Pen G per ml resulted in theaccumulation in the culture medium of material that ab-sorbed strongly at 225 nm. Much of the UV-absorbingmaterial was of a molecular size that eluted from a SephadexG-100 column with a Kd of ca. 0.1 (Fig. 1A, peak I). Whencells were incubated in this medium containing [14C]glucoseor [3H]glucose (see Fig. 1), [14C]lysine or [3H]lysine, or

['4C]alanine (or a combination of these), material eluting

I01'10CM1qcw

Kd

600B. o

400- 0 0%

E

*0~~~~~~~~~~~~~~~~~~~200L jDf\ .

01~~~~~~~~~~~.0 0.25 0.50 0.75 1.0

KdFIG. 1. (A) Gel filtration on Sephadex G-100 of polymers secret-

ed by the Lyt-14 strain of S. faecium ATCC 9790 when incubated inwall medium in the presence of Pen G. Shown are two experiments.In experiment 1 (dashed line), the supernatant medium from an 800-ml culture incubated for 1 h at 37°C (containing [per milliliter] 10 ,ugof Pen G and 0.88 mg of cells [dry weight] from an exponential-phaseculture pregrown in S broth) was heat inactivated, partially dialyzed(3,500-molecular-weight retention dialysis tubing), concentrated,and filtered on a Sephadex G-100 column. Elution of material fromthe column with water was monitored by absorption of each fraction(2.5 ml) at 225 nm (A225) (A). In experiment 2 (solid lines), a 50-mIculture containing (per milliliter) 10 ,ug of Pen G and 1.0 mg of cells(dry weight) from an exponential-phase culture pregrown in S brothcontaining [14C]glucose (3.6 ,uCi/mmol, 0.4 ,uCi/ml) (0) was incubat-ed for 1 h at 37°C with [3H]glucose (35.7 p.Ci/mmol, 2 ,uCi/ml) (0)present in the wall medium as new label with s-peptidoglycanisolated as described above. (B) Sephadex G-100 elution profile of s-peptidoglycan secreted by the parental S. faecium strain. Thesupernatant medium from a 50-ml culture incubated 1 h at 30°C(containing [per milliliter] 10 ,ug of Pen G and 0.88 mg of cells[dry weight] from an exponential-phase culture pregrown in S broth,[14C]glucose [35.7 jiCi/mmol, 2 ,uCi/ml] [0], and [3H]lysine [188.4p.Ci/pmol, 3 ,uCi/ml] [0]) was heat inactivated, partially dialyzed(3,500-molecular-weight retention dialysis tubing), and filtered on aSephadex G-100 column (37 by 2.5 cm) at 4°C. The concentrations ofunlabeled glucose and lysine in the medium were reduced to 1/10 thenormal amount to facilitate incorporation of the label.

with a Kd of about 0.1 contained the respective radiolabel(s).Material present in peak I (Fig. 1A) was found to behydrolyzed to low-molecular-weight products (dialyzablethrough 5,000-molecular-weight retention dialysis tubing) byHEWL. With paper chromatography (solvent 1), peak Imaterial remained at the origin, whereas the products of

VOL. 159, 1984


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/


TABLE 1. Factors influencing the optimum synthesis of soluble peptidoglycans by strain Lyt-14"

Culture

Harvestand ~~~~~~~~~~~~~~~Cellturbidity at TotalExpt warveshteanCell wash Wall medium culture harvest" (mg cellular Yield ofExpt wash temp procedure composition vol of cellular mass" (mg/g of cells)

(liters) dry wt per (g)ml)

la 32 None MWMd' 2 0.2 0.39 0.36lb 32 Water MWM 2 0.2 0.39 1.04lc 32 NaPO4' MWM 2 0.2 0.39 6.44

2a 4 NaPO4" MWM 6 0.2 1.17 1.052b 32 NaPO4t MWM 6 0.2 1.17 6.97

3a 32 NaPO4' MWM 3 0.16 0.47 7.23b 32 NaPO4' MWM + NaPO4 3 0.16 0.47 9.8

" All cultures of strain Lyt-14 were grown in S broth at 37C and, after harvest and wash (as indicated in the table), were incubated in wall medium containing 50,ug of Pen G per ml at 37C for 1 h.

Determined by absorption at 675 nm.Estimated at time of harvest from complex medium.

" MWM, Modified wall medium described in the text.80 mM, pH 6.8.0.3 M, pH 6.8.

HEWL hydrolysis of peak I migrated away from the origin,yielding a series of discrete spots (37, 43). In contrast,several proteinases (e.g., trypsin, chymotrypsin, and subtili-sin BPN' at 100 ,ug/ml) failed to hydrolyze material in peak I.

Thus, based on data from similar experiments with otherorganisms (37-39), these initial experiments were interpretedas indicating that strain Lyt-14 (and strain Aut-3), whenincubated in wall medium with Pen G, secreted high-molecu-lar-weight s-peptidoglycan.Source of s-peptidoglycan. An exponential-phase culture of

strain Lyt-14 was grown for more than seven generations inthe presence of ['4C]glucose to assure equilibration of theradiolabeled precursor (43). Cells were harvested, washed,and suspended in wall medium containing Pen G and[3H]glucose. After 1 h at 37°C, the supernatant was removedand the s-peptidoglycan isolated as described above. Materi-al in the high-molecular-weight peak (Kd of 0.1 on thiscolumn; Fig. 1A, peak I) contained only 3H and no detect-able 14C, indicating that the polymer recovered from thesupernatant medium was newly synthesized and not pro-duced by loss (e.g., by turnover) of previously assembled

cell wall. Peak II (Fig. IA) contains unincorporated label(the amount depended on the completeness of dialysis) pluslow-molecular-weight peptidoglycan fragments.

Production of s-peptidoglycan by the parental strain of S.faecium. In contrast to the autolysis-defective strains of S.faeciimn, Lyt-14 and Aut-3, incubation of the autolysis-prone, parental strain of S. faeciurn in wall medium contain-ing [14C]glucose, [3H]lysine, and 10 ,ug of Pen G per ml for1 h resulted in the production of material that eluted fromSephadex G-100 with a K,1 of greater than 0.6 (Fig. 1B).HEWL, but not proteinases, hydrolyzed this low-molecular-weight material to substances that migrated from the originon paper chromatography in solvent 1. Thus, this productwas considered to be low-molecular-weight s-peptidoglycanfragments resulting from hydrolysis by the endogenousmuramidase activity present in the parental strain. Shorterincubations (20 or 40 min in wall medium) also failed to showthe production of material with a Kd of less than 0.6.

Optimal conditions for production of s-peptidoglycan. Avariety of variables, including incubation time, Pen G con-centration, and temperature and washing procedures during

TABLE 2. Amino acid analyses of strains Lyt-14 and Aut-3 s-peptidoglycans"Amt and concn of amino acid in s-peptidoglycan from:

Amino acid Lyt-14, preparation 1 Lyt-14. preparation 2 Aut-3

MlrMolar Molar~Lmol/mg Moa Vtmol/mg Molar [mlm oa

ratio' ratio ,umollmg ratio

Aspartic acid 0.49 0.60 0.47 0.58 0.51 0.60Glutamic acid 0.81 1.00 0.81 1.00 0.85 1.00Alanine 2.50 3.09 1.24 1.53 2.67 3.18Lysine 0.73 0.90 0.87 1.07 0.86 1.01Serine 0.03 0.02 0.06 0.07 0.05 0.06Tyrosine <0.01 <0.01 <0.01Leucine <0.01 <0.01 <0.01Isoleucine <0.01 <0.01 <0.01Histidine 0.02 0.02 0.03 0.04 0.03 0.04Methionine <0.01 <0.01 <0.01Arginine 0.05 0.06 0.12 0.14 0.09 0.11Glycine 0.07 0.08 0.13 0.16 0.13 0.15

" Preparation 1 was obtained from a culture exposed to 50 ,ug of Pen G per ml in wall medium (plus 0.3 M sodium phosphate, pH 6.8), and preparation 2 was ob-tained from a culture exposed to 5 ,ug of Pen G per ml. Aut-3 s-peptidoglycan was obtained from wall medium (plus 0.3 M sodium phosphate, pH 6.8) containing 50p.g of Pen G per ml. Values are averages of duplicate or triplicate determinations.

b Molar ratios to glutamic acid.

J. BACTERIOL.


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/


harvesting of cells and their transfer from growth medium towall medium (Table 1 and Fig. 2), were examined with theview of increasing s-peptidoglycan yield. In summary, thefactors that proved to be important were: (i) washing thecells from an exponential-phase culture with sodium phos-phate, pH 6.8 (0.04 to 0.2 M); (ii) harvesting and washing at32 to 37°C rather than at 4°C; (iii) the presence of a highconcentration of sodium phosphate (0.3 M) in the wallmedium; and (iv) the time of incubation and Pen G concen-tration (Fig. 2). Pen G concentrations, from 10 to 100 ,ug/ml,resulted in decreased incorporation of [14C]glucose into acid-insoluble material (Fig. 2B) and increased production of 4C-s-peptidoglycan (Fig. 2A). A very high Pen G concentration(1 mg/ml) resulted in both little incorporation of 14C intoacid-insoluble material and an intermediate level of s-pepti-doglycan secretion. At this high Pen G concentration, somecellular lysis (decrease in turbidity) of both lysis-defectivestrains was observed. At the optimal Pen G concentrations(10 to 100 ,ug/ml), optimal yields of polymer were obtained atca. 1 h; longer incubations resulted in decreased amounts ofpolymer, presumably due to its hydrolysis by the residualautolysin present in these strains (45, 53).Other factors that proved important (data not shown)

were: (i) a reasonably low turbidity (less than 200 p.g ofcellular dry weight per ml) of the growing cultures at the timeof harvest and (ii) the heat inactivation step. This latter stepnot only prevented endogenous hydrolysis of the polymerbut resulted in an easily removed small precipitate (presum-ably protein). Omission of this step resulted in a decreasedyield of polymer and an increased amount of protein in peakI on the Sephadex G-100 column.

It is of considerable interest to note that s-peptidoglycanproduced in the presence of 50 p.g of Pen G per ml (Lyt-14,preparation 1, and Aut-3; Table 2) contained 3 alanines perglutamate, whereas the s-peptidoglycan produced in thepresence of a lower Pen G concentration (5 p.g/ml; Lyt-14,preparation 2; Table 2) contained only 1.5 alanines perglutamate. This difference was attributed to inhibition of theDD-carboxypeptidase of this species at high Pen G concen-trations (5).Chemical composition of the s-peptidoglycan. Amino acid

analyses of the soluble polymers of strains Lyt-14 and Aut-3demonstrated the presence of the same amino acids previ-ously identified in cell walls of Lyt-14 (4) and Aut-3 (53). Theamino acids were present in approximately the same molarratios, and amino acids found primarily in proteins were notpresent in significant amounts (Table 2). Peaks at the elutionpositions expected for muramic acid and glucosamine con-tained ninhydrin-reactive material, but the amino sugarswere not quantified because of the strong acid hydrolysisconditions used. After milder hydrolysis in 3 N HCl, 95°C,for 4 h, the Morgan-Elson assay (17) indicated the presenceof 1.84 and 2.03 ,umol of amino sugars per mg in the strainLyt-14 (preparation 1) and strain Aut-3 s-peptidoglycans,respectively (Table 3). In each of the preparations, the sumof amino acids and amino sugars accounted for 98.3 and99.7% of the dry weight of the s-peptidoglycan from strainsLyt-14 (preparation 1) and Aut-3, respectively.The s-peptidoglycan preparations contained only trace

amounts of methylpentose, neutral sugars, or phosphorus(Table 3), indicating that the polymers contained little non-peptidoglycan wall polymers, such as teichoic acid andpolysaccharides. Analysis of reducing groups before andafter acid hydrolysis or complete enzymatic hydrolysis withthe SF muramidase indicated that the glycan chain lengths ofthe s-peptidoglycans were ca. 42 to 46 and 45 to 48 disaccha-

0.~~~~~~~0B.

60-

40

00

0 30 60 90 120 O/NMINUTES

FIG. 2. The effect of Pen G on the incorporation of [14C]glucoseinto s-peptidoglycan (A) and into trichloroacetic acid-insolublematerial by strain Lyt-14 (B). A culture (1 liter) of strain Lyt-14 wasgrown to 0.18 mg of cellular dry weight per ml and was harvested,washed, and suspended in 220 ml of wall medium. This 220-mlculture was divided into six 35-ml portions, to which variousconcentrations of Pen G (from 0 to 1 mg/ml of wall medium) wereadded. After 5 min at 37°C, [14C]glucose (10 ,uCi/mmol, 0.57 ,uCi/ml)was added to each of the six flasks. At intervals, duplicate 1-mlsamples were taken, and cell pellets and supernatants were separat-ed by centrifugation (12,000 x g, 5 min, 4°C) in 1.8-ml conicalpolypropylene tubes. Supernatants were heat inactivated (100°C, 5min) and stored at 0°C. Cell pellets were quick-frozen in the conicaltubes in an ethanol-dry ice mixture and stored at -20°C until subjectto cold trichloroacetic acid precipitation as described in the text.The duplicate samples of the supernatant were dialyzed extensively(8,000-molecular-weight retention dialysis tubing) six times against 4liters of water at 4°C and were lyophilized and reconstituted in 1 mlof water, of which 250 p.1 was counted (A). The pellets were subjectto cold trichloroacetic acid preparation, and the total insolubleradioactivity incorporated into each sample was determined (B).Pen G (micrograms per milliliter): Control, none (A); 0.1 (0); 1 (0);10 (0); 100 (-); and 1,000 (A).

ride-peptide monomer units long for the polymers of strainsLyt-14 and Aut-3, respectively (Table 4). Comparisons ofMorgan-Elson-positive groups before and after acid hydroly-sis or complete enzymatic hydrolysis with the SF murami-dase (Table 4) were consistent with these estimates. Thenumber average molecular size of the s-peptidoglycans wasdetermined by gel filtration over an AcA 34 column (Table

VOL. 159, 1984


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/


TABLE 3. Chemical analyses of strain Lyt-14 and strain Aut-3 s-peptidoglycans

Peptidoglycan from:

Component Lyt-14 (preparation 1) Aut-3

,umol/mg Molar ratio" ,umol/mg Molar ratio

Amino sugarsAfter acid hydrolysis 2.03 1.02 1.84 0.97After SF muramidase 0.99 N.A." 0.92 N.A.hydrolysis

Reducing endsBefore hydrolysis 0.022 0.01 0.02 0.01After acid hydrolysis 1.99 1.00 1.9 1.00After SF muramidase 0.90 N.A. 0.92 N.A.hydrolysis

Phosphorus 0.012 0.01 0.017 0.01

Neutral sugars 0.013 0.01 0.017 0.01

Methylpentose 0.013 0.01 0.015 0.01

Molar ratio of the indicated component compared with reducing groupsdetermined after acid hydrolysis. Values are averages of duplicate determina-tions.

" N.A.. Not applicable.

4). These estimates of molecular sizes of the s-peptidogly-cans were consistent with the chemical analyses (Tables 3and 4).

Reducing terminus of the s-peptidoglycan. The reducingterminus of the strain Lyt-14 s-peptidoglycan, as repre-sentative of the soluble polymers, was determined to bemuramic acid by identification of the sugar alcohol deriva-tive after [3H]NaBH4 reduction, acid hydrolysis (6 N HCI,12 h, 100°C), and paper chromatography (Table 5). Treatmentof the strain Lyt-14 s-peptidoglycan with [3H]NaBH4 beforeand after complete hydrolysis to disaccharide-peptide mono-mers with the muramidase showed that the reducing termi-nus of the s-peptidoglycan was muramic acid. In addition,the products of SF muramidase hydrolysis of s-peptidogly-can had muramic acid at their reducing termini and showedan ca. 41-fold increase in reducing groups (Table 5). Theseresults are consistent with the hydrolysis of essentially all

susceptible glycosidic bonds in the polymer. Furthermore,this chain length determination is in good agreement withboth the physical and other chemical determinations of chainlength of the strain Lyt-14 s-peptidoglycan (Table 4).

s-Peptidoglycan production and size of the polymer. Studiesof the kinetics of production of s-peptidoglycan and itsmolecular size, with time of incubation (Fig. 3), showed thatalthough the amount of polymer produced increased withincubation time, the apparent molecular weight of the s-

peptidoglycan produced was the same after 30 s as it was

after the 20-min incubation period.

DISCUSSION

Incubation of either of two phenotypically autolysis-defec-tive strains of S. jaecium (Lyt-14 and Aut-3) with Pen Gduring nongrowth conditions in a chemically defined medi-um (48) which supports peptidoglycan synthesis (wall thick-ening) but not protein synthesis resulted in the secretion of s-

peptidoglycan into the medium. The s-peptidoglycans fromstrains Lyt-14 and Aut-3 absorbed UV light strongly at 220 to225 nm and weakly at 260 and 280 nm and were eluted near

the void volume (K,d = 0.05 to 0.2) on a Sephadex G-100column (Fig. LA). The parental strain of S. fraecium was

found to produce only low-molecular-weight polymers (Fig.1B), presumably due to the action of its active, endogenousautolytic system. Growing protoplasts of S. faecium alsosecrete low-molecular-weight peptidoglycan fragments (39,40). Inhibition of protein synthesis with tetracycline (1 pLg/ml) and adsorption of extracellular autolysin by addedsodium docecyl sulfate-inactivated cell walls showed thatthe low-molecular-weight peptidoglycan fragments secretedby the protoplasts resulted from muramidase hydrolysis oflonger (peptido)glycan chains (41). In the experiments pre-

sented here, this problem was circumvented by the use ofautolysis-defective mutants (4, 53).The maximum amount of s-peptidoglycan produced by

these strains was ca. 20% of the amount expected (asinsoluble cell wall peptidoglycan) of exponentially growing

cultures of strain Lyt-14. However, this amount of s-pepti-doglycan was comparable to the amount of insoluble wallpeptidoglycan estimated to be made by the parental strain ofS. faetcium when incubated in the same wall medium withoutPen G (48). Over 98% of the s-peptidoglycans of both theLyt-14 and Aut-3 strains were accounted for by wall amino

TABLE 4. Calculation of glycan chain length and apparent molecular weight of s-peptidoglycans"Calculated glycan chain length" Apparent mol wt of strain:

Formula or source of calculations in strain:Lyt-14 Aut-3 Lyt-14 Aut-3

Reducing groups after acid hydrolysis/reducing 45.5 48.0 50,050 52,800groups before hydrolysis

Reducing groups after complete hydrolysis by the 40.5 46.0 44,550 50,600SF muramidase/reducing groups before hydrolysis

Morgan-Elson-positive groups after acid hydrolysis/ 46.0 46.0 50,600 50,600reducing groups before hydrolysis

Morgan-Elson-positive groups after complete hydro- 45.0 46.0 49,500 51,150lysis by the SF muramidase/reducing groups be-fore hydrolysis

AVG 44.0 ± 2" 46.5 ± 1 48,675 ± 2,100 51,150 + 1,000

Gel filtratione 46,000 ± 4,300 53,000 ± 5,700" s-Peptidoglycan preparations secreted in the presence of 50 p.g of Pen G per ml and purified as described in the text.bIn disaccharide-peptide units. Calculation of ratio of values such as those shown in Table 3.Calculation based on approximate molecular weight of a dissacharide-peptide monomer unit of 1,100.

dAverage deviation.Gel filtration on AcA 34.

J. BACTERIOL.


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/


TABLE 5. End group analysis of strain Lyt-14 s-peptidoglycanby detection and identification of sugar alcohol'

Sugar alcohol present kcpm Totalkcpmb

Before SF muramidase hydrolysisMuramicitol 108 2.46Glucosaminitol ND"

After complete SF muramidase hydrolysisMuramicitol 4,400 100Glucosaminitol NDa s-Peptidoglycan was exhaustively reduced by [3H]NaBH4 as described in

the text, and the products were identified after acid hydrolysis. Paperchromatography standards migrated in solvent 2 as follows: solvent front (Rf)of 14.5 cm has a muramicitol peak at 4.5 cm (Rf = 0.31) and a glucosaminitolpeak at 1.5 cm (Rf = 0.10).

b Based on the sum of all counts per minute recovered on paper chromato-graphs after SF muramidase hydrolysis, reduction with [3H]NaBH4, and acidhydrolysis.

c ND, None detected.

acids and amino sugars (Tables 2 and 3). The molar ratios ofamino acids were very similar to those for walls of the threestrains (4, 16, 52, 53), except for an ca. 30% lower amount ofaspartate in all three s-peptidoglycans and a higher amountof alanine in the s-peptidoglycans made at high penicillinconcentrations. Analyses of the products of HEWL and SFmuramidase hydrolysis of the s-peptidoglycan showed noevidence for the presence of peptide cross-links (2). Thepresence of only traces of phosphorus, neutral sugars, andmethylpentose indicates the absence of accessory wall poly-mers (Table 3). The s-peptidoglycans produced by all thestrains of S. faecium examined contained substituents on allof their muramic acid residues based on chromatographicanalyses of hydrolysis products and on amino acid analyses(Table 2). Thus, S. faecium s-peptidoglycans differ from, M.luteus s-peptidoglycan that contains, at most, 50% peptide-substituted N-acetylmuramic acid residues (30), presumablydue to the action of an endogenous amidase (23). Thereducing terminus of the strain Lyt-14 s-peptidoglycan wasidentified as muramic acid (Table 5) and is similar to thatreported for other gram-positive organisms secreting thispolymer and, thus, may represent a general structure of thegrowing chain of newly made peptidoglycan. Nonreducingmuramic acid end groups, such as the anhydromuramic acidends found in Escherichia coli (22, 54), were not detected.

All of the data obtained are consistent with the synthesisand secretion by the autolysis-defective strains of (peptido)-glycan chains containing 42 to 48 disaccharide-peptidemonomer units of 46,000 to 54,000 molecular weight. Therelative uniformity of glycan length of the secreted s-pepti-doglycans, even after incubation of cells for only 30 s in wallmedium, contrasts with the high level of heterogeneity ofglycan chain lengths observed in insoluble walls of a varietyof bacteria (37). These results are similar to the relativelyuniform glycan chains (12 to 14 disaccharide-peptide units)extracted from Bacillus subtilis by Fuchs-Cleveland andGilvarg (15) and to the synthesis in vitro of teichoic acidchains containing 22 to 24 ribitol units by preparations ofStreptococcus lactis 13 (60). The latter investigators pro-posed that a specific glycan chain length could be critical fordissociation of growing chains from the synthesizing com-plex.

Although probably premature, it is tempting to proposethat the secreted glycan chains are representative of anoligomeric biosynthetic intermediate that is later processed

by peptidoglycan hydrolases (e.g., endogenous murami-dases) after incorporation into the wall via transpeptidation(see reference 46 for a review). However, it should berecalled that these glycan chains were recovered from asupernatant medium of bacterial cells engaged in wall thick-ening rather than wall surface enlargement. Although grossdifferences in chemical composition and structure of thick-ened and normal walls have not been recorded, smalldifferences in detail and chemical structure could easilyexist. Further data will be required to detertnine the role, ifany, of such glycan oligomers in cell wall assembly.The apparent absence of nonpeptidoglycan wall polymers

covalently linked to the soluble (peptido)glycan chains, andeven the absence of phosphorus through which such poly-mers are frequently linked to carbon 6 of N-acetylmuramicacid, is consistent with observations of s-peptidoglycansecretion in other penicillin-inhibited systems (25, 29, 59, 61,65) and with the secretion of peptidoglycan fragments bygrowing protoplasts of S. faecium (39-41) and reverting L-forms of Bacillus licheniformis (9). In the system describedhere and in growing protoplasts of S. faecium (39), noevidence for the synthesis and secretion of wall teichoic acidwas obtained. Even the use of [14C]glucose or [3H]glucoseprecursors failed to reveal the presence of radiolabel inoligomeric products that could not be identified as s-peptido-glycan or peptidoglycan fragments. These results suggestthat synthesis of teichoic acids and other nonpeptidoglycanwall polymers may depend on the incorporation of (peptido)-glycan chains into the cell wall. Also, all of these data areconsistent with the covalent linkage of nonpeptidoglycanwall polymers to the peptidoglycan after the synthesis of the(peptido)glycan chains and, perhaps with or after their

0

%-k

Ea.

l 0, .

0 5 10 15 20MINUTES

FIG. 3. Time course of production (and molecular size) of s-peptidoglycan by strain Lyt-14 in wall medium. Shown is theapproximate molecular size of strain Lyt-14 s-peptidoglycan deter-mined by gel filtration on a calibrated Sephadex G-200 column (c)and the rate of production of s-peptidoglycan of molecular sizegreater than 8,000 (0). Strain Lyt-14 was grown in S broth (100 ml)to 0.18 mg of cellular dry weight per ml, harvested by filtration,washed with 25 ml of prewarmed (37°C) 0.08 M sodium phosphate(pH 6.8), and incubated in 20 ml of wall medium containing 0.3 Msodium phosphate (pH 6.8) and 50 ,ug of Pen G and [14C]glucose(1.43 mCi/mmnol, 8 ,uCi/ml) per ml. Unlabeled glucose in the wallmedium was reduced 10-fold to facilitate 14C incorporation. Atintervals, 1.8-ml samples were removed, the cells were separated bycentrifugation (12,000 x g, 5 min), and the supernatant was removedand heat inactivated at 100°C for 7 min. Samples were stored at-20'C until applied to a Sephadex G-200 column. Counts areexpressed in disintegrations per minute of high-molecular-weight s-peptidoglycan. Approximate molecular size is the number averageof high-molecular-weight s-peptidoglycan.

VOL. 159, 1984


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/


incorporation into insoluble walls, usually via 3-lactam-sensitive transpeptidation reactions.The characterization of the secreted cell wall material as

an uncross-linked, soluble peptidoglycan of a specific, highmolecular weight and free of accessory ligands represents adefined substrate which has been used to examine the kineticproperties of the muramidase of S. faecium as described inthe accompanying paper (2).

ACKNOWLEDGMENTS

We thank D. Mirelman for sending us M. luteus NCTC 2665 andfor his advice during the early phases of this project; A. R. Zeiger,J. K. Hoober, and R. Millington for amino acid analysis; and H.Goldstein for design and implementation of computer analysisprograms. We are grateful to W. Wong, T. McDowell, and V. L.Schramm for discussions and for their comments on the manuscriptand to G. Harvey for his help in preparing the manuscript.

This work was supported by Public Health Service research grantAl 05044 from the National Institutes of Health. J.F.B. was support-ed by Public Health Service National Research Service Awardtraining grant Al 07101.

LITERATURE CITED

1. Akrigg, A., and S. R. Ayad. 1970. Studies on the competence-inducing factors of Bacillus subtilis. Biochem. J. 117:397-403.

2. Barrett, J. F., V. L. Schramm, and G. D. Shockman. 1984.Hydrolysis of soluble, linear, uncross-linked peptidoglycans byendogenous bacterial N-acetylmuramoylhydrolases. J. Bacte-riol. 159:520-526.

3. Chatterjee, A. N., W. Wong, F. E. Young, and R. W. Gilpin.1976. Isolation and characterization of a mutant of Staphylococ-cus aureus deficient in autolytic activity. J. Bacteriol. 125:961-967.

4. Cornett, J. B., B. E. Redman, and G. D. Shockman. 1978.Autolytic defective mutant of Streptococcus faec alis. J. Bacte-riol. 133:631-640.

5. Coyette, J., J.-M. Ghuysen, F. Binot, P. Adriaens, B. Meess-chaert, and H. Vanderhaeghe. 1977. Interactions between ,B-lactam ahtibiotics and isolated members of Streptococcus fae-calis ATCC 9790. Eur. J. Biochem. 75:231-239.

6. Daneo-Moore, L., and G. D. Shockman. 1977. The bacterial cellsurface in growth and division, p. 597-713. In G. Poste andG. L. Nicholson (ed.), Cell surface reviews, vol. 4. Elsevier/North Holland Biomedical Press, Amsterdam.

7. Dische, Z., and L. B. Shettles. 1948. A specific color reaction ofmethyl pentoses and a spectrophotometric micromethod fortheir determination. J. Biol. Chem. 175:595-603.

8. Dubois, M., K. A. Gilles, J. K. Hamilton, P. A. Rebers, and F.Smith. 1956. Colormetric method for determination of sugarsand related substances. Anal. Chem. 28:350-356.

9. Elliott, T. S. J., J. B. Ward, and H. J. Rogers. 1975. Formationof cell wall polymers by reverting protoplasts of Bacill/uslicheniformis. J. Bacteriol. 124:623-632.

10. Fan, D. P. 1970. Autolysin(s) of Bacillus subtilis as dechainingenzyme. J. Bacteriol. 103:494-499.

11. Fan, D. P., M. C. Pelvit, and W. P. Cunningham. 1972.Structural difference between walls from ends and sides of therod-shaped bacterium Bacillus suibtilis. J. Bacteriol. 109:1266-1272.

12. Fein, J. E. 1979. Possible involvement of bacterial autolyticenzymes in flagellar morphogenesis. J. Bacteriol. 137:933-946.

13. Fein, J. E., and H. J. Rogers. 1976. Autolytic enzyme-deficientmutants of Bacillus subtilis 168. J. Bacteriol. 127:1427-1442.

14. Forsberg, C. W., and H. J. Rogers. 1974. Characterization ofBacillus licheniformis 6346 mutants which have altered lyticenzyme activities. J. Bacteriol. 118:358-368.

15. Fuchs-Cleveland, E., and C. Gilvarg. 1976. Oligomeric interme-diate in peptidoglycan biosynthesis in Bacillus megate-iumn.Proc. Natl. Acad. Sci. U.S.A. 73:4200-4204.

16. Ghuysen, J.-M., E. Bricas, M. Leyh-Bouille, M. Lache, and

G. D. Shockman. 1967. The peptide N-(L-alanyl-D-isogluta-minyl)-N'-(D-isoasparaginyl)-L-lysyl-D-alanine and the disac-charide N-acetylglucosaminyl-,3-1,4,-N-acetylmuramic acid incell wall peptidoglycan of Streptococcus faecalis strain ATCC9790. Biochemistry 6:2607-2619.

17. Ghuysen, J.-M., D. J. Tipper, and J. L. Strominger. 1966.Enzymes that degrade bacterial cell walls. Methods Enzymol.8:685-699.

18. Higgins, M. L., and G. D. Shockman. 1970. Model for cell wallgrowth of Streptococcus faecalis. J. Bacteriol. 101:643-648.

19. Higgins, M. L., and G. D. Shockman. 1971. Procaryotic celldivision with respect to wall and membranes. Crit. Rev. Micro-biol. 1:29-72.

20. Higgins, M. L., and G. D. Shockman. 1976. Study of a cycle ofcell wall assembly in Streptococcus faecalis by three-dimen-sional reconstructions of thin sections of cells. J. Bacteriol.127:1346-1358.

21. Hinks, R. P., L. Daneo-Moore, and G. D. Shockman. 1978.Relationship between cellular autolytic activity, peptidoglycansynthesis, septation, and the cell cycle in synchronized popula-tions of Streptococcus faecium. J. Bacteriol. 134:1074-1080.

22. Holtje, J. V., D. Mirelman, N. Sharon, and U. Schwarz. 1975.Novel type of murein transglycosylase in Escherichia coli. J.Bacteriol. 124:1067-1076.

23. Jansen, S. E., and J. N. Canipbell. 1976. Amidase activityinvolved in peptidoglycan biosynthesis in membranes of Micro-coccus luteus (sodonensis). J. Bacteriol. 127:319-326.

24. Kawamura, T., and G. D. Shockman. 1983. Purification andsome properties of the endogenous, autolytic N-acetylmura-moylhydrolase of Streptococcus faecium, a bacterial glycoen-zyme. J. Biol. Chem. 258:9514-9521.

25. Keglevic, D., B. Ladesic, 0. Hadzija, J. Tomasic, Z. Valinger, M.Pokorny, and R. Naumski. 1974. Isolation and study of thecomposition of a peptidoglycan complex excreted by the biotin-requiring mutant of Brevibacterium dii'aricatuin NRRL-2311 inthe presence of penicillin. Eur. J. Biochem. 42:389-400.

26. Lowry, 0. H., N. R. Roberts, K. Y. Leiner, M.-L. Wu, and A. L.Farr. 1954. The quantitative histochemistry of brain. 1. Chemi-cal methods. J. Biol. Chem. 207:1-17.

27. Mauck, J., L. Chan, and L. Glaser. 1971. Turnover of the cellwall of gram-positive bacteria. J. Biol. Chem. 246:1820-1827.

28. McLean, C., D. A. Werner, and D. Aminoff. 1973. Quantitativedetermination of reducing sugars, oligosaccharides, and glyco-proteins with 3H-borohydride. Anal. Biochem. 55:72-84.

29. Mirelman, D., R. Bracha, and N. Sharon. 1974. Penicillin-induced secretion of a soluble, uncross-linked peptidoglycan byMicrococcus luteus cells. Biochemistry 13:5045-5053.

30. Mirelman, D., and N. Sharon. 1967. Isolation and study of thechemical structure of low molecular weight glycopeptides fromMicrococcus /ysodeikticus cell wall. J. Biol. Chem. 242:3414-3427.

31. Munson, R. S., and L. Glaser. 1981. Teichoic acids and peptido-glycan assembly in gram-positive organisms, p. 91-122. In V.Ginsburg and P. Robbins (ed.), Biology of carbohydrates, vol.1. John Wiley & Sons, Inc., New York.

32. Park, J. T., and M. J. Johnson. 1949. A submicrodeterminationof glucose. J. Biol. Chem. 181:149-151.

33. Pelzer, H. 1963. Muropeptidhydrolasen in Escheric hia coli B. Z.Naturforsch. Teil B 18:950-956.

34. Pooley, H. M., J. M. Porres-Juan, and G. D. Shockman. 1970.Dissociation of an autolytic enzyme-cell wall complex by treat-ment with unusually high concentrations of salt. Biochem.Biophys. Res. Commun. 38:1134-1140.

35. Ranhand, J. M., C. G. Leonard, and R. M. Cole. 1971. Autolyticactivity associated with competent group H streptococci. J.Bacteriol. 106:257-268.

36. Rogers, H. J. 1970. Bacterial growth and the cell envelope.Bacteriol. Rev. 34:194-214.

37. Rogers, H. J., H. R. Perkins, and J. B. Ward. 1980. Microbialcell walls and membranes, p. 437-460. Chapman and Hall,London.

38. Rogers, H. J., H. M. Pooley, P. F. Thurman, and C. Taylor.1974. Wall and membrane growth in bacilli and their mutants.

J. BACTERIOL.


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/


Ann. Microbiol. (Paris) 125B:135-147.39. Rosenthal, R. S., D. Jungkind, L. Daneo-Moore, and G. D.

Shockman. 1975. Evidence for the synthesis of soluble peptido-glycan fragments by protoplasts of Streptococcus faecalis. J.Bacteriol. 124:398-409.

40. Rosenthal, R. S., and G. D. Shockman. 1975. Characterization ofthe presumed peptide cross-links in the soluble peptidoglycanfragments synthesized by protoplasts of Streptococcus faecalis.J. Bacteriol. 124:410-418.

41. Rosenthal, R. S., and G. D. Shockman. 1975. Synthesis ofpeptidoglycan in the form of soluble glycan chains by growingprotoplasts (autoplasts) of Streptococcus faecalis. J. Bacteriol.124:419-423.

42. Roth, G. S., G. D. Shockman, and L. Daneo-Moore. 1971.Balanced macromolecular biosynthesis in "protoplasts" ofStreptococcus faecalis. J. Bacteriol. 105:710-717.

43. Rupley, J. A. 1964. The hydrolysis of chitin by concentratedhydrochloric acid, and the preparation of low-molecular-weightsubstrates for lysozyme. Biochim. Biophys. Acta 83:245-255.

44. Seto, H., and A. Tomasz. 1975. Protoplast formation and leakageof intramembrane cell components: induction by the compe-tence activator substance of pneumococci. J. Bacteriol. 121:344-353.

45. Shockman, G. D. 1965. Symposium on the fine structure andreplication of bacteria and their parts. IV. Unbalanced cell-wallsynthesis: autolysis and cell wall thickening. Bacteriol. Rev.29:345-358.

46. Shockman, G. D., and J. F. Barrett. 1983. Structure, functionand assembly of cell walls of gram-positive bacteria. Annu. Rev.Microbiol. 37:501-527.

47. Shockman, G. D., and M. C. Cheney. 1969. Autolytic enzymesystem of Streptococcus faecalis. V. Nature of the autolysin-cell wall complex and its relationship to properties of theautolytic enzyme of Streptococcus faecalis. J. Bacteriol. 98:1199-1207.

48. Shockman, G. D., M. J. Conover, J. J. Kolb, L. S. Riley, and G.Toennies. 1961. Nutritional requirements for bacterial cell wallsynthesis. J. Bacteriol. 81:44-50.

49. Shockman, G. D., L. Daneo-Moore, and M. L. Higgins. 1974.Problems of cell wall and membrane growth, enlargement anddivision. Ann. N.Y. Acad. Sci. 235:161-197.

50. Shockman, G. D., L. Daneo-Moore, T. McDowell, and W. Wong.1981. Function and structure of cell wall-its importance in lifeand death of bacteria, p. 31-66. In M. Salton and G. D.Shockman (ed.), P-Lactam antibiotics. Academic Press, Inc.,New York.

51. Shockman, G. D., L. Daneo-Moore, T. D. McDowell, and W.Wong. 1982. The relationship between inhibition of cell wallsynthesis and bacterial lethality, p. 202-220. In R. B. Morin and

M. Gorman (ed.), The chemistry and biology of 3-lactamantibiotics, vol. 3, Academic Press, Inc., New York.

52. Shockman, G. D., J. S. Thompson, and M. J. Conover. 1967. Theautolytic enzyme system of Streptococcus faecalis. lll. Partialcharacterization of the autolysin and its substrate. Biochemistry6:1054-1065.

53. Shungu, D. L., J. B. Cornett, and G. D. Shockman. 1979.Morphological and physiological study of autolytic-defectiveStreptococcus faecium strains. J. Bacteriol. 138:598-608.

54. Taylor, A., B. C. Das, and J. van Heijenoort. 1975. Bacterial-cell-wall peptidolgycan fragments produced by phage X or Vi Itendolysin and containing 1,6-anhydro-N-acetylmuramic acid.Eur. J. Biochem. 53:47-54.

55. Thompson, J. S., and G. D. Shockman. 1968. A modification ofthe Park and Johnson reducing sugar determination suitable forthe assay of insoluble material. Anal. Biochem. 22:260-268.

56. Toennies, G., and D. L. Gallant. 1949. The relation betweenphotometric turbidity and bacterial concentration. Growth 13:7-20.

57. Tomasz, A. 1968. Biological consequences of the replacement ofcholine by ethanolamine in the cell walls of pneumococci. Proc.Natl. Acad. Sci. U.S.A. 59:86-93.

58. Tomasz, A. 1979. The mechanism of the irreversible antimicro-bial effects of penicillins: how the beta-lactam antibiotics killand lyse bacteria. Annu. Rev. Microbiol. 33:345-358.

59. Tynecka, Z., and J. B. Ward. 1975. The inhibition of cross-linking by benzylpenicillin and cephaloridine in vivho accompa-nied by the formation of soluble peptidoglycan. Biochem. J.146:253-267.

60. Watkinson, R. J., H. Hussey, and J. Baddiley. 1971. Shared lipidphosphate carrier in the biosynthesis of teichoic acids andpeptidoglycan. Nature New Biol. (London) 229:57-59.

61. Waxman, D. J., W. Yu, and J. L. Strominger. 1980. Linear,uncross-linked peptidoglycan secreted by penicillin-treated Ba-cillus subtilis. J. Biol. Chem. 255:11577-11587.

62. Wenzel, M., H.-P. Lenk, and E. Schutte. 1962. Herstellug vontri-(N-acetyl)-chitotriose-[3H] und deren Spaltung durch Lyso-zyme. Z. Physiol. Chem. 327:13-20.

63. Wong, W., F. E. Young, and A. N. Chatterjee. 1974. Regulationof bacterial cell walls: turnover of cell wall in Streptococcusaureus. J. Bacteriol. 120:837-843.

64. Young, F. E., D. J. Tipper, and J. L. Strominger. 1964.Autolysis of cell walls of Bacillus subtilis: mechanism andpossible relationship to competence. J. Biol. Chem. 239:PC3600-PC3602.

65. Zeiger, A. L., W. Wong, A. N. Chatterjee, F. E. Young, andC. U. Tuazon. 1982. Evidence for the secretion of solublepeptidoglycans by clinical isolates of Staphylococcus alureus.Infect. Immun. 37:1112-1118.

VOL. 159, 1984


http://jb.asm.org/

Dow

nloaded from

http://jb.asm.org/

Isolation and Characterization of Soluble Peptidoglycan from ...

Documents

Transcript of Isolation and Characterization of Soluble Peptidoglycan from ...