An invitro system for the biosynthesis of spore cortex peptidoglycan.

An in vitro system for the biosynthesis of spore cortex peptidoglycan Deptrr!t~ieti!yf'Biologic~c11Sciet~i,e.t,Brock Utiii~er.sicy,Sf.Cothorit~es,0111. , Coticrclcr LZS 3A I

Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by CONCORDIA UNIV on 12/10/14 For personal use only.

Accepted March 31, 1977 PEARCE,S. M. 1977. An in vitro system for the biosynthesis of spore cortex peptidoglycan. Can. J. Microbiol. 23: 964-974. A simple crude system for the incorporation of tritiated diaminopimelic acid ('H-DAP) into a polymer with characteristics of spore cortical peptidoglycan has been obtained from cells of Bocillrr.~cere1t.s var. trlcsti, harvested and disrupted in a French pressure cell, at the time of late cortex formation (sporulation stage 5 ) . The small fraction of whole cells remaining in the homogenate were not responsible for the observed incorporation. T h e radioactive product was sensitive t o digestion by lysozyme a s is coltex formed in vivo. Preparations obtained at the same time of sporulation, from a mutant unable t o form cortex. were unable to incorporate 'H-DAP into peptidoglycan. However. homogenates prepared at the time of germ cell wall formation (early stage 4) from both parent and cortexless mutant produced radioactive peptidoglycan which was more resistant t o lysozyme as is germ cell wall produced in vivo in this species. Bacitracin and vancomycin inhibited incorporation of 'H-DAP into peptidoglycan by over 90% in both cell wall and cortical preparations. Methicillin caused a striking inhibition of 'H-DAP incorporation into peptidoglycan by the cortical system in contrast t o its almost total lackof inhibition of the cell wall or germ cell wall systems. Over 90% of the DAP incorporated was the expected mesoisomer. )H-DAP-labelled lipid intermediate was produced by the system. Cross-linking of newly synthesized material was observed. This in vitro systemappears satisfactory for the further study of some aspects of the biosynthesis of cortical peptidoglycan. PEARCE,S. M. 1977. An in vitro system for the biosynthesis of spore cortex peptidoglycan. Can. J. Microbiol. 23: 964-974. Nous avons obtenu. en utilisant des cellules d e Btrci1lrr.s cererrs var. crlesri recoltkes au moment d e la formation tardive du cortex et dtsintegrees par la presse d e French, un systeme simple permettant I'incorporation d'acide diaminopimelique tritie ('H-DAP) dans un polymere possedant les caracttristiques du peptidoglycan cortical de la spore (stade 5 d e la sporulation). Le petit nombre d e cellules non desintegries persistant dans I'homogenat n'etait pas responsable de I'incorporation observee. Comme le cortex synthetise in vivo, le produit radioactif e s t sensible h la digestion lysozymique. Des preparations obtenues au meme stade de sporulation et a partir d'un mutant incapable d e former un cortex ont demontre une incapacite d'incorporer le 'H-DAP au peptidoglycan. Cependant, les homogenats plipares au stade de debut d e formation d e la paroi cellulaire (stade 4 d e la sporulation) a partir d e la souche mere et d'un mutant sans cortex p r o d ~ ~ i s e un n t peptidoglycan radioactif d'une resistance accrue au lysozyme tout c o m m e lajeune paroi cellulaire produite par cette espece in vivo. La bacitracine et la vancomycine inhibent I'incorporation d e W - D A P au peptidoglycan dans une proportion d'au-deli d e 90% pour les preparations de parois cellulaires ainsi que d e cortex. La methicilline entraine une forte inhibition d e I'incorporation d e 'H-DAP au peptidoglycan par le systeme cortical; ce phenomene contraste x. tnP.70 constitue avec I'absence presque complete d'inhibition d e s systemes p a r i e t a ~ ~ L'isomere au-deli d e 90% du D A P incorpore. Le systeme produit un intermediaire lipidique marque par le 'H-DAP. Nous avons note que le materiel nouvellement synthttise etait ponte. C e systeme in vitro nous apparait satisfaisant pour I'etude plus approfondie d e certains aspects d e la biosynthese d u peptidoglycan cortical. [Traduit p a r le journal]

Illtroduction Extensive studies on bacterial cell wall peptidoglycans have led to a comparatively thorough understanding of the structure and biosynthesis of the basic unit (27). Recent research has been focussed more on the ter~ninalsteps and assembly of the three-dimensional polymer (13, 14, 23, 29, 30, 31). Little is known, however, of the bio-

synthesis of bacterial spore cortical peptidoglycan, a polymer similar to cell wall peptidoglycan, yet differing in several unique features. Wickus et (11. (37) and Imae and Strominer (8) did corc with relate the appearance of ~ n u r a ~ n ilactam cortex synthesis, and showed that the assay of ~nuramiclactarn is a good measure o f cortex content.

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The cortex, a major ~iiorphologicallayer in mature spores, is essential for the maintenance of the spore properties of heat resistance, refractility, and dormancy (7, 15, 20). Its basic structure consists of repeating units of N-acetylglucosaniine-N-acetyl muramic acid with peptides substituted on some of the muramic residues. Whereas in cell wall peptidoglycans, every muramic residue has a peptide sidechain most of which are cross-linked to other peptides, in cortical peptidoglycan up to 5 0 x of the muramic residues are substituted by a unique spore component, the lactam ring, and up to 3 0 x by L-alanine only (34, 35). Thus cortical peptidoglycan is ~iiuchless extensively cross-linked, is consequently inore pliable than that of cell wall, and has a greater excess of carboxyl groups (32, 35). These properties suit the concept of the cortex as an ion exchanger capable of swelling and contracting ( 7 , 15). Biosynthesis of cortical peptidoglycan should be basically a similar process to that of cell wall peptidoglycan since, in both cases, soluble precursors must cross a liiembrane barrier t o the site of polymerization. An in vitro system for the synthesis of cortical peptidoglycan would allow the efficient addition and utilization of precursors, the ~nanipulationof the process necessary for a detailed understanding of it, and a comparison of it and cell wall biosynthesis. One inajor problem in obtaining such a system lies in determining whether the peptidoglycan product is truly of a cortical nature, since a sporulating cell has three different peptidoglycan structuresthe cell wall proper, the germ cell wall (formed before cortex), and the spore cortex. This paper describes a crude in vitro systeiii in which a peptidoglycan product having some of the characteristics of spore cortical peptidoglycan is formed.

Methods and Materials Orgor~irr)rsotrd Cultiootiotr A non-crvstal-formine strain of Bricilllrs cn.ei1.s var. olesfi designated A(-) w a s used (4). Both stocks were maintained on plates of nutrient agar (Difco), the mutant being subcultured every 7 days. For sporulation in liquid medium, the techniques and medium used were basically those of Young and Fitz-James (38), and are described in full elsewhere ( 1 8). Microscopy Phase-contrast microscopy was used for routine exam~nationof sporulating c~~ltur'es. An estiniation of the n degree of synchrony and the stage of s p o r ~ ~ l a t i owas

made by counting the percentage of forespores that had hecome phase white. This is a somcwhat subjective procedure but does allow comparison of experiments. Greater than 90% of forespores whitened within 1 h and about 9 9 z sporulation was observed. Numbers of whole cells before and after disruption were counted directly with a Neubauer haemocytometer. Samples were prepared for electron microscopy by the method of Kellenberger cf (11. (1 1 ) with some niodifications as described previously (I 9). A Phillips 200 electron niicroscope was used. 1'ift.o Sysfe177for Pcplirlaglyctrrr S~'tll/resis Cultures at the appropriate stage of growth or sporulation were centrifuged and the pellet suspended in t h volume of 0.1 iL1 tris (hydroxymethyl) aminomethane (Tris) HCI, pH 7.5. Cells were disrupted by passage twice through a French pressure cell at 12000 1b/sq in pressure, and the resulting homogenate checked routinely by phase niicroscopy for extent of breakage. Aliquots of the homogenate (usually 2.0 ml) were added to s ~ n a l lflasks containing a niixture with components to give the followi~ig final conccntrations: D,L-alanine, lo-' IM: D-glutamic acid, lo-' M; L-lysine, IM; N-acctyl glucosanline, lo-, itl; MgCI,, lo-' M, MnCI,, M : uridine 5'-triphosphate (UTP), M; adenosine 5'-triphosphate (ATP), 5 x lo-' &I; and 3H-DAP, 0.5 ~ ~ C i i rat n la concentration of 1.6 x IM. The incubation was stopped usually after 30 t o 60 niin at 30°C, by t h e addition of 4 0 z trichloroacetic acid (TCA) t o a final concentration of 5%. FOI- most experinients, the peptidoglycan was purified froni the TCA precipitates by the method of Park and Hancock (17), and aliquots were placed o n 0 . 4 5 - ~ n l membrane filters (Millipore Corp.), washed thoroughly, and counted for radioactivity. Itr

Cl~c~t~tic~nl At1o1~~si.s Protein was estimated by the method of Lowry cf (11. (1 2). Hydrolysis of extracted peptidoglycan was carried out in 6 A' HCI for 16 h a t 105 C. High-voltage electrophoresrs was done on Whatman No. 3 MM paper, a t 40 V,cm for 1 h using a buffer made of 12.4 nil of fo~.niicacid and 43.5 ml of glacial acetic acid per litre. The following chromatography solvents were used. Solvent I-rl~ethanol : water : 12 N HCI : pyridine (80: 17.5:2.0:10 v/v, ref. 22); solvent 11-isobutyric acid : 1 M NH,OH (5:3 v/v, ref. 25); solvent 111-butanol saturated NH,OH; solvent IV-1-aniyl alcohol s a t ~ ~ r a t e d with with 0.1 A4 phthalate buffer, p H 6.0 (36); solvent Vbenzyl alcohol : chloroforni : methanol : water : concentrated NH,OH (15:15:23:10:1 vjv, modified from ref. 6).

1z

Lysozytlle Set~.~ifi~.il}~ The pellet resulting from centrifugation of the TCA precipitate from incubation mixtures as described above, was washed twice with water to reniove TCA and was suspended in 5.0 ml of 0.01 M Tris-HCI buffer, pH 7.5. Aliauots were filtered before a n d after incubation at 37°C with lysozyrne (100 pg/rnl). The amount of rad~oactivity remaining in insoluble material was expressed a s a percentage of t h a t present before lysozyme addition.


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CAN. J. MICROBII

Exfrocrion ofLipid Itzfertlietlinte Cells were harvested and disrupted in &th volume of 0.1 M Tris I-ICI buffer, pH 7.5. The incubation mixture was as described with the 3H-DAP concentration increased to 2 pCi/ml, and total volumes increased to 10 to 20 ml. After 15 min the lipids were extracted with an equal volun~eof butanol : 6 N pyridine acetate, pH 4.2 (2:l v/v, ref. 28). The butanol phase was washed thoroughly with water containing non-radioactive DAP (100 pg/ml), flash-evaporated at 37"C, dissolved in chloroform:methanol (1 :1, v/v), and spotted on Whatman 3 M M chromatography paper. The chromatogram was developed for 16 h in solvent 11.

lb/sq. in. pressure, the resulting homogenate contained some whole forespores, fragments of forespores, sporangial membranes and cell wall, and spore coats as well as all soluble cell components (Fig. 1). Direct counting of whole cells before and after disruption showed that less than 0.1% of the original cell number was unbroken. This figure may have been a n overestimation in that cells with minor breaks in cell wall and membrane (as in Fig. 1) would have appeared as whole cells in the light microscope. The degree of disruption of cell structure was Dinitr.opherlylofiot~ Hydrolysates of 3H-DAP-labellcd pcptidoglycan were less than in homogenates prepared by sonication treated with fluorodinitrobenzene (FDNB) and the dini- or by disruption of cells in a Mickle tissue distrophenylated (DNP-) derivatives extracted into ether, integrator or a Braun disintegrator. These latter by the method of Ghuysen et nl. (6). The DNP-derivatives were scparated by thin-layer chromatography on methods were totally unsatisfactory in producing silica gel using solvent 111. An aliquot of 3H-DAP to homogenates capable of the in vitro synthesis of which unlabelled DAP was added as carrier was also 3H-DAP-labelled peptidoglycan in the test dinitrophenylated and chromatographed as a marker. Bands corresponding to the di-DNP-derivative of meso- system described, as less than 2% incorporation DAP, and the Di-DNP-derivatives of D,D- and L,L-DAP of 3H-DAP was observed relative to French together were scraped off and eluted and counted in press preparations. Addition of 10% glycerol o r phase combining system (PCS):xylene (2:l) scintillation 10 m M M g 2 + t o the smash buffer did not enfluid. hance activity of the preparations. A suitable quench curve was prepared and used to Incorporation of 3H-DAP into peptidoglycan correct for colour quenching by the yellow DNPby preparations of A(-) disrupted during mid t o derivatives. For estimation of cross-linking extracted 3H-DAP late cortex formation (70% of forespores phase labelled peptidoglycan was dinitrophenylated directly white) and added to the test system was linear using the same method. After ether extraction, the DNPpeptidoglycan was centrifuged and washed three times with time t o 120 min, and linear with protein with 70% ethanol and three times with water before acid concentration, at least to 8 mg/ml. The optimum hydrolysis was done. Chromatography was done in temperature was about 30°C. The optimum p H solvent V. was 7.5 (Fig. 2). High-voltage electrophoresis of The mono-2,4-dinitrophenol derivativc of unlabelled hydrolysed purified peptidoglycan showed that D A P (mono-DNP-DAP) was prepared according to over 99% of the tritium label remained as Fraenkel-Conl-at et 01. ( 5 ) , and purified by thin-layer chronlatography in solvent V. Mono-DNP-3H-DAP was 3H-DAP. also PI-epared and purified. The total system was compared t o incubation mixtures lacking specific components. There was Scintillntion Corrt~/it~g Membrane filters or paper chromatography strips no absolute dependence o n any one component, were placed in scintillation vials containing 10 ml of the although omission of each reduced incorporation scintillant; 4 g of 2,5-diphenyloxazole per litre and 0.1 g (Table 1). The omission of ATP gave the most of 1,4-bis-[2-(5-phenyloxazolyl)]-benzene per litre in scintillation grade toluene. Eluted samples from thin- variable results, the reduction in activity varying layer chromatography were added to PSC:xylene (2:l). from 50 t o 80% of the activity of the complete system. Omission of all additives other than the Cl~enricnls u-e-Diaminopimelic acid-T(G) dihydrochloride and 3H-DAP from the test system only reduced the incorporation of 3H-DAP by 60% compared t o PCS (Phase Combining System) were obtained from Amersham-Searle; lysozyme and bacitracin were ob- the complete system. This was not surprising in tained from Sigma Chemical Company; vancomycin that the homogenate contained all soluble pool from Eli Lilly Company; niethicillin was a gift from materials and enzymes present at the time of Dr. P. C. Fitz-James. disruption. Addition of phosphoenol pyruvate (PEP) and reduced pyridine nucleotides did not Results increase activity indicating that formation of muramic acid was not-a limiting factor. The total Basic Sysf en? When cells in sporulation stages 4 or 5 were amount of added DAP incorporated in the comdisrupted in a French Pressure cell a t 12 000 plete system as described was about 0.1 t o 0.2

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FIG.1. Electron micrograph of a preparation of A(-) disrupted in a French Pressure cell during midsporulation. The sporangial cell wall (ov) and membranes (tn) are broken. Some forespores (fs) remain reasonably intact while others are more extensivelv damaged. Fragments of cell wall. membrane vesicles, and other cell debris are seen in the preparation. 32 400.-

x

nmol/mg protein per hour. This amount of D A P could be increased substantially by increasing the concentration of D A P in the assay by the addition of unlabelled D A P , but this lowered the A ~ in specific activity of the 3 ~ - a n~d resulted

less radioactivity being incorporated. T h e figure quoted for D A P incorporation is based o n the specific activity of the added DAP, a n d thus is the minimum amount incorporated since the extent o f dilution by pool D A P from the dis-

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TABLE 2. Effect of addition of known numbers of intact cells to a homogenate on the incorporation of 'H-DAP into peptidoglycan

% whole cells


addeda

cpmb

'Percentage of whole cells added is expressed a s a percentage of the number of whole cells present in the in vitro assay due to the small number of unbroken cells remaining in t h e hornogenate after disruption. *This refers t o the "H-DAP counts present in a 1/10 aliquot o f purified peptidoglycan extracted after 40 min of incubation i n the usual in vitro assay system.

pH FIG.2. The effect of pH on the incorporation of 3HDAP into peptidoglycan by preparations of A(-) disrupted during midcortex formation. Results are expressed as a percentage of the amount of 3H-DAP incorporated at the optimal pH of 7.5.

E

TABLE1. Effect of omission of specific components from the incubation mixture, on the incorporation of 'H-DAP into peptidoglycan by preparations of A(-) cells harvested in midsporulation I

% incorporation of 'H-DAP Complete system - ATP - Mg2 -Mn2+ - Alanine - Glutamic acid - N-acetyl glucosamine -A11 components +

rupted cells was not determined. The extent of incorporation of 3H-DAP by whole cells under the conditions of the in vitro assay was determined and found to be less than 10 times that incorporated by an equivalent number of disrupted cells. Since the French Press fractured over 99.9% of cells in the in vitro assay, the 0.1% of whole cells would at most contribute l z of the observed incorporation of 3H-DAP into peptidoglycan. T o check this further, known amounts of whole cells varying from 20 to 220% of the number of whole cells present in homogenates (as determined by direct counting) were added to homogenates in the in vitro assay system. N o effect was seen o n the rate of incor-

10

30 MINUTES

60

FIG.3. Digestion by lysozyme (100 pg/ml) of the 3HDAP-labclled product of in vitro systems for the incorporation of cortical peptidoglycan, 0 ; cell wall peptidoglycan, A ;and germ cell wall peptidoglycan, B.

poration of 3H-DAP (Table 2), again indicating negligible contribution by whole cells. Lysozyme Sensitit'ity of the Product Since in B. cereus strains, cell wall and germ cell wall are lysozyme resistant while the cortex is sensitive (33) lysozyme sensitivity of the radioactive products of the in vitro system was investigated. Homogenates of A(-) cells prepared during vegetative growth, germ cell wall synthesis (after engulfment), and cortex synthesis (70% phase white) were used. The cortical product was almost completely digested while the cell wall and germ cell wall products were only partially digested at a slower rate (Fig. 3). The increased extent of digestion of the germ cell wall material compared to cell wall product may have been due to the presence of some cortical material due to slight asynchrony of the cultures.

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D

25 -

5 10 15 pg VANCOMYCIN /mg PROTEIN

20

FIG, 4, Effect of concentration of vancomycin on incorporation of 3H-DAP into peptidoglycan both in vitro and in vivo under conditions of the in vitro assay. Cortical peptidoglycan in vitro, 0 ; cell wall peptidoglycan in vitro, D;cortical peptidoglycan in vivo, 0.

Inhibition by Cell Wall Inhibitors The effects of known inhibitors of cell wall synthesis on the formation of peptidoglycan polymers in this in vitro system were investigated using homogenates of A(-) cells in midlogarithmic growth and midsporulation (70% phase white). A range of concentration of bacitracin, vancomycin, and methicillin (rather than penicillin since B. cereus produces a potent penicillinase, (21) was used, and ability of homogenates to incorporate 3 H - ~ A assayed. P Results are plotted as percentage of activity of uninhibited control against micrograms of antibiotic per milligram of protein (Figs. 4,5, 6). Between 2.5 and 3 pg of protein were used per millilitre in all assays. Sensitivity to bacitracin appeared similar for cell wall and cortex synthesis; a slight difference was seen in sensitivity to vancomycin. Methicillin gave considerable inhibition of cortical peptidoglycan synthesis but incorporation of 3H-DAP into cell wall peptidoglycan was virtually unaffected a t low concentrations. In vitro incorporation of 3H-DAP into germ cell wall peptidoglycan showed no sensitivity to methicillin. These effects contrasted with the results obtained with whole cells. Whole cells in midsporulation (6570% phase white) were harvested, washed, and suspended in Ath volume of 0.1 M Tris-HCI, p H 7.5, as for the preparation of homogenate, and added to the in vitro incubation mixture with the same range of inhibitors as used above. N o inhibition of 3H-DAP

0.5

1.0

~g BAGITRAGIN /rng

1.5 PROTEIN

2.0

'

FIG. 5. Effect of concentration of bacitracin on incorporation of 3H-DAP into peptidoglycan both in vitroand in vivo under conditions of the in vitro assay. Cortical peptidoglycan in vitro, 0 ; cell wall peptidoglycan in vitro, D;cortical peptidoglycan in vivo, 0.

I

10 20 30 pg METHlClLLlN /rng PROTEIN

40

FIG.6. Effect of concentration of methicillin on incorporation of 3H-DAP into peptidoglycan both in vitro and in vivo under conditions of the in vitro assay. Cortical peptidoglycan in vitro, 0 ; cell wall peptidoglycan in vitro, A ; germ cell wall peptidoglycan in vitro, B; cortical peptidoglycan in vivo, 0.

incorporation by whole cells was seen (Figs. 4, 596). Mode of Insertion of 'H-DAP Two chromatography methods were used to determine which isomer of DAP was incorporated into polymer by this in vitro system. Paper chromatography according t o Rhuland et al. in solvent I , of the hydrolysate products from


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extracted 3H-DAP-labelled peptidoglycan was carried out. The chromatograms were cut into 1-cm strips and counted for radioactivity. Unlabelled D A P (mixture of D,D-, L,L-, and meso(D,L-) isomers) was used as a marker and detected by ninhydrin spray. In this system L,L-DAP moves ahead of the u,u- and mesoisomers which run together. No. L,L-DAP was detected. T o distinguish between the D- and the mesoisomers, the di-DNP derivatives of the hydrolysis products were made and chromatographed first by silicic acid thin-layer chromatography in solvent 111, and also by paper chromatography in solvent IV. Over 95% of the di-DNP-3H-DAP product was the meso-isomer. This is the isomer always found in the pentapeptide side chains of cell wall peptidoglycans which contain DAP. This finding of meso-DAP as the major isomer of DAP incorporated and the inhibition of incorporation of label into polymer by vancomycin and bacitracin indicated that DAP was incorporated into polymer via lipid-linked intermediates similar t o those found in cell wall synthesizing systems. An attempt was made to isolate these intermediates, using the documented methods for extraction of cell wall lipid intermediates as described in the methods, and chromatography in solvent 11. A peak of radioactive material with an Rf of about 0.9 (0.860.93) was detected as well as a small amount of free 3H-DAP (about Rf 0.4) which had not been removed by the washing procedure. Cell wall lipid intermediates have an RJ of 0.9 in this system (28). An approximate figure for the amount of incorporation of D A P into lipid intermediate was calculated to be about 0.3 pmol DAP/mg protein after 15 min of incubation. Subsequent experiments showed that labelling of lipid intermediate had reached this level by 5 min of incubation and remained approximately constant for a t least the following 15 min, while incorporation of 3H-DAP into peptidoglycan increased. Bacitracin (5 pg/mg protein) decreased the amount of label in lipid intermediate by over 5 0 x . Vancomycin (15 pg/mg protein) increased the amount of label by up to 100%. The action of these inhibitors was not further quantitated, nor was the labelled lipid material further characterized except for the demonstration that the tritium remained as 3H-DAP.

Cross-linking of Incorporated DAP Extracted 3H-DAP-labelled peptidoglycan synthesized by preparations of A(-) cells disrupted during cortex formation was treated with FDNB. Uncross-linked D A P would react with this reagent whereas cross-linked DAP would be unaffected. O n hydrolysis the mono-DNP derivative of D A P and free D A P would be released. After thin-layer chromatography in solvent V, comparison of the amount of tritium label in the mono-DNP-DAP band t o that in the free D A P band gave a measure of the extent of cross-linking. Several controls were needed. With each experiment, a sample of pure monoDNP-3H-DAP of known specific activity was hydrolysed with a n amount of unlabelled peptidoglycan equivalent to the amount used in the test hydrolysis vials, and treated as for the test samples so as to obtain a correction factor for thc extent of breakdown of the DNP-DAP derivative on hydrolysis. This figure was about 10% for total breakdown with about 3% appearing in the free D A P band. The amount of radioactivity found in the yellow bands eluted from the silicic acid was corrected for colour quenching by the construction of a suitable quench curve, In two experiments the figure obtained for the extent of cross-linking of D A P was 44.5% 1.5%. Thus this crude system is capable of crosslinking D A P presumably by transpeptidation. Discussion Preparations which are capable of the incorporation of 3H-DAP into insoluble peptidoglycan have been obtained from sporulating cells of B. cereus var. alesti. These preparations appear suitable for a study of some aspects of the biosynthesis of cortical peptidoglycan. The methods of mechanical breakage of cells and of purification of membrane fragments used in earlier studies on cell wall peptidoglycan synthesis (16, 28) severely disrupt cell membranes and probably cause substantial alterations in the physical orientation of the membrane, in enzyme activity, and in the three-dimensional relationship of the membrane to cell wall components. More recently, crude cell wall-membrane preparations and toluenized cells have bcen used in attempts to understand more fully the terminal steps in biosynthesis of peptidoglycan and the mode of assembly of the complete three-dimensional wall structure (13, 23, 24, 30). The French

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pressure cell method of breakage used here is much less disruptive than the usual mechanical breakage methods. Furthermore, the major permeability barrier to precursors added externally, that is, the sporangial plasma membrane, is broken allowing the direct addition of precursors and inhibitors, and thus, the efficient utilization of these and the manipulation of the biosynthetic system. The amount of 'H-DAP incorporated into peptidoglycan by the basic assay system was about 10% of the added 'H-DAP per hour (about 0.1 to 0.2 nmol added 'H-DAP/mg protein per hour). That this incorporation is not due simply to the less than 0.1% of whole cells in the preparation is shown by several lines of evidence. The experiments on a number of whole cells present in the homogenates, on incorporation of 'HDAP by whole cells under conditions of the in vitro assay, and on the effect of addition of known numbers of whole cells to homogenates, all indicate that the contribution of whole cells to the observed incorporation of 3H-DAP into peptidoglycan is small and probably less than 1%. Furthermore ATP which cannot enier whole cells stimulated incorporation by up to 100% (Table 1). Finally, bacitracin, vancomycin, and methicillin markedly inhibited in vitro cortical peptidoglycan synthesis while giving no inhibition of incorporation of 'H-DAP into peptidoglycan by whole cells in midcortex formation (Figs. 4, 5, 6). This is not surprising in that in whole cells the inhibitors must pass the sporangial cell wall and plasma membrane, and in the case of methicillin the outer forespore membrane before reaching their site of action. In disrupted cells, they are able to act directly. Calculations from figures (Pearce, unpublished) on the pool size of N-acetyl amino sugar esters (mainly uridine 5'-diphosphate UDPsugar precursors of peptidoglycan synthesis (26)) during sporulation indicate that the 'H-DAP may be added to preexisting incomplete nucleotide precursors, that is, total de nouo synthesis of the UDP-monosaccharide pentapeptide is not necessarily taking place. The failure of phosphoenolpyruvate (PEP) or reduced pyridine nucleotides t o stimulate the incorporation of 3H-DAP into peptidoglycan is in agreement with this. D A P has been shown to be incorporated into peptidoglycan in cell wall - synthesizing systems

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by two mechanisms (36): the general mechanism of initial incorporation of meso-DAP into UDPmonosaccharide pentapeptide followed by transfer to polymer via lipid-linked intermediates, found in all organisms containing D A P in their peptidoglycan, and the less common transpeptidation reaction found in B. megaterium in which D A P (both meso- and D,D-forms) is added directly to polymer. That at least 90% if the 'H-DAP incorporated by the present system occurred by the first mechanism rather than by transpeptidation has been demonstrated. Over 95% of the incorporated D A P was of the mesoform with the remainder apparently in the D,Dform (no L,L- was incorporated), although the radioactive DAP added was of almost equal amounts of meso- and D,D- plus L,L-forms. The incorporation of 'H-DAP was accompanied by the formation of 3H-DAP-labelled material which chromatographed in a similar fashion t o cell wall lipid intermediates. The incorporation was inhibited by over 90% by the cell wall inhibitors bacitracin and vancomycin, neither of which should inhibit transpeptidase activity. About 0.3 pmol 3H-DAP per milligram of protein was incorporated into lipid intermediate material after 5 min of incubation, and remained relatively constant for a further 15 min. This figure was again based on the specific activity of the added 3H-DAP and thus may be a n underestimate due to dilution of the 'H-DAP by DAP of cell pools. Many rounds of transfer of precursors via lipid intermediate must have occurred in the usual 1-h incubation assay to account for the amount of label which appeared in insoluble product. The amount of label in this lipid-like material was increased by the addition of vancomycin and decreased by the addition of bacitracin, as is expected from the known action of these inhibitors (1, 2, 25). No attempt was made to maximize the extent of inhibition by these antibiotics. The crude system described here appears capable of some of the terminal steps of peptidoglycan synthesis since preliminary experiments showed that about 45% of the incorporated 3H-DAP was cross-linked. This figure is similar to that reported by Warth and Strominger (35) for cortical peptidoglycan of Bacillus subti[is spores. I t does not give any indication of the number of amino sugars carrying a peptide side chain. Whether the other terminal modifications


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of peptidoglycan which occur in cortex (muramic lactam formation. occurrence of muramic residues substituted by L-ala only) are carried out by this preparation is not known. This in vitro svstem does exhibit considerable specificity in the nature of the polymer made. The products formed showed different sensitivities to lysozyme as expected, since cortex in this organism is lysozyme-sensitive whereas both cell and germ cell wall are more resistant (33). Inhibition by bacitracin was similar for both cortical product and cell wall product; vancomycin inhibited incorporation of 3H-DAP into cortical peptidoglycan to a slightly greater extent than cell wall. However, inhibition by methicillin showed the most dramatic difference. Whereas the in vitro incorporation of 3H-DAP into cell wall or germ cell wall peptidoglycans was little affected, methicillin caused a dramatic inhibition (50% at 15 pg methicillin/mg protein) of 3H-DAP into cortical peptidoglycan. This striking difference may reflect differences in the three-dimensional assembly of the peptidoglycan particularly with respect to the role of the transpeptidase which is penicillin-sensitive (9). Early studies on cell wall synthesis by purified membrane preparations showed no inhibition by penicillin on the incorporation of soluble peptidoglycan precursors (1, 3, 10, 27) while in more recent studies with crude cell wall - membrane preparations incorporation of peptidoglycan precursors has been shown to be substantially inhibited by penicillins (13, 14, 30). It seems likely that either transpeptidation is the major mechanism for the joining of new peptidoglycan t o existing cell wall, or that there is some inhibition of g ~ y ~ o s y ~ a t if ion peptide crosslinking is inhibited. The results presented here simply indicate a further difference between the synthesis and assembly of cortical and cell wall ieptidoglycans in B. cereus var. alesti. Experimerits to determine whether soluble uncrosslinked material is synthesized in the presence of methicillin have not been done as vet. A further example of specificity in this in vitro system comes from previous studies on a 'lutant derived from *(-) and to form a cortex. In vivo studies showed that incorporation of 3 H - D ~ into ~ cortical peptidoglycan occurred with little overlap of germ cell wall synthesis in B. cereus var. alesti, a n d that A(-)l did not incorporate 3H-DAP into peptidoglycan during the period of normal

cortex formation (19). Homogenates of the cortexless mutant were tested in the in vitro system described in this paper (18). Briefly, homogenates of A(-)l prepared from cells at the time of normal cortex formation (70% phase white) were unable to incorporate 3H-DAP into peptidoglycan, while homogenates of A(-)l prepared at the time of germ cell wall formation (after engulfment) incorporated similar amounts of DAP as the parent, A(-). These in vitro results paralleled those found in vivo and showed that no reactivation of the germ cell wall or cell wall - synthesizing systems was occurring in vitro. This paper thus presents preliminary characterization of an in vitro system of peptidoglycan synthesis which seems to have sufficient specificity to allow further study of some aspects of cortex synthesis. Some interesting questions that should be answered are: Is muramic lactam formed? Is a tetrapeptide liberated leaving L-ala-substituted muramic acid residues? What is the direction of chain growth? What is the reason for the striking methicillin sensitivity?

Acknowledgements This study was supported by a National Research Council of Canada Grant. The author thanks Dr. P. C. Fitz-James, University of Western Ontario, for carrying out the electron microscopy. It was in his laboratory that the author initiated this study. The assistance of Susie Fung and Alf Kaethler is appreciated.

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.

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