Biosynthesis of Peptidoglycan in the One Million Molecular Weight Range by Membrane Preparations from

Abstract Peptidoglycan made in vitro by membrane preparations of Bacillus megaterium had a buoyant density in CsCl different from that of native cell wall. This newly made material was not covalently attached to substantial amounts of previously formed cell material such as wall. From sedimentation velocity centrifugation, the molecular weight of much of the in vitro product was found to be greater than 6 x 106. Peptidoglycan made in the presence of penicillin had smaller molecular weights suggesting that transpeptidation was in part responsible for the large size of the product. With reaction conditions in which transpeptidation was presumed to have occurred, release of alsnine was observed. This release was inhibited by penicillin.

made in vifro by membrane preparations of Bacillus megaterium had a buoyant density in CsCl different from that of native cell wall. This newly made material was not covalently attached to substantial amounts of previously formed cell material such as wall. From sedimentation velocity centrifugation, the molecular weight of much of the in vitro product was found to be greater than 6 X 106. Peptidoglycan made in the presence of penicillin had smaller molecular weights suggesting that transpeptidation was in part responsible for the large size of the product. With reaction conditions in which transpeptidation was presumed to have occurred, release of aknine was observed. This release was inhibited by penicillin.
It has been reported that Bacillus megaterium membrane preparations can synthesize peptidoglycan cross-linked in two different patterns (1). One of these patterns involves the removal of the terminal alanine from a peptide side chain and the attachment of the rest of the peptide to a diaminopimelic acid residue of another peptide.
Since this type of transpeptidation is also seen in many other organisms, experiments were undertaken to study the physical properties of the peptidoglycan made in vitro under conditions where only such cross-linking could occur. It was found that B. megaterium membrane preparations could make a substantial amount of cross-linked peptidoglycan of molecular weight greater than approximately 6 x 106. This material was not linked to previously existing cell wall.
One method used to study peptidoglycan made in c&o is State Kidney Disease Institute, Albany, New York 12208. 5 To whom correspondence should be addressed at the Department of Genetics and Cell Biology. electron microscopy. However, for this technique to be useful, it is advantageous for the product to be as large as possible.
In order to make a large product resembling closely that made in uioo, the isolated membranes should obviously be damaged as little as possible. Therefore, a new method of cell breakage was employed for preparing membranes.
In this method bacteria were cut cleanly in two in the frozen state. This procedure was presumed to be more gentle than techniques employed previously. EXPERIMENTAL  The suspension was boiled 5 min, and the debris was removed by 5-min centrifugation at 12,ooO X g. The supernatant was mixed with 169 ~1 of 2 N NaOH and boiled again for 5 min. The solution was recentrifuged at 12,66~1 x g for 5 min to remove any precipitated material. The residues removed by these centrifugation steps were pooled, and the amount of radioactivity was determined in a liouid scintillation counter.
The final super-preparation described above was added to 4.8 ml of CsCl in Buffer B (0.1 M KCI-0.1 M Tris, pH 8.6) with a refractive index for the sodium D line at 25" (~~21) of 1.383 as measured in an Abbe refractometer.
CsCl solutions with this refractive index have a density of 1.522 g per cc (8).
When desired, B. megaterium walls prepared as described above were added to the CsCl solution to provide a density marker. The usual method of addition was to sediment the walls by centrifugation and resuspend the pellet in the CsCl solution.
All samples of peptidoglycan made in vitro together with density marker walls were thoroughly mixed with the entire volume of CsCl solution required to form the densitv gradients prior to centrifugation.
All-gradients were centrifuged at 186,066 X 9 (SW 50.1 rotor at 39.609 mm in a Sninco ultracentrifune) for 16 to I8 hours at 20".
Fractions were collected through the-hypodermic syringe needle used to puncture the bottoms of the tubes. Sometimes the refractive index was measured for the samples, and the corresponding densities were obtained from a table of densities (8).
The position of the marker walls was obtained by diluting the fractions with water and reading the turbidity at 540 nm in a spectrophotometer.
For measurements of radioactivity in the peptidoglycan, drops from the gradient were collected directly onto Whatman No. 3MM filter paper. These gradient fractions on pieces of filter paper were immersed in a large volume of 5yc trichloroacetic acid. After extensive mixing, the solution of trichloroacetic acid containing the extracted acid-soluble peptidoglycan precursors was discarded. The papers were washed two more times with large volumes of 5% trichloroacetic acid in a similar manner.
The papers were dried and counted in a liquid scintillation counter. After all the gradient fractions had been collected, the bottom rounded portion of the centrifuge tube including any materials which collected as a pellet during centrifugation was removed with a sharp blade and counted by liquid scintillation.
The radioactivity in this fraction was plotted in the position of fraction zero in the gradient profiles.

Sucrose Gradient
Analysis-Fifteen microliters of concentrated HCI were added to the synthetic reactions prepared for centrifugation by sarkosyl and NaOH extraction.
The pH was checked with pH paper and was adjusted to pH 7 to 8 using small amounts of2NHClor2NNaOH.
A This last technique has not been generally used and will be discussed in some detail.
Electron micrographs of cells prepared by freeze-etching show that most cell structures remain intact after this treatment (10). Therefore, a pellet of cells was rapidly frozen in liquid nitrogen and ground to a fine powder with a mortar and pestle. As the pellet was fractured into small pieces, bacteria in the pellet were broken cleanly in two.
Since very few of the fracture lines passed through bacteria, only natant having a volume of approximately 200 pl was used for-the approximatelv 10% of the bacteria were broken. The frozen --__ ." centrifugation analysis. CsCl Equilibrium Density Gradient Centrifugation-The 290-~1 and fractured pellet was then thawed, and a membrane preparation was made from the few cells that were broken. Since r The abbreviations used are: UDP-GlcNAc, UDP-N-acetyl-n-the pellet was always frozen during breakage, the membranes glucosamine; UDP-MurNAc-Ala-Glu-Dap-Ala-Ala, UDP-N-ace-from broken cells could be damaged mechanically only at the tyl-D-muramyl-L-alanyl -D -r-glutamyl -meso -diaminopimelyl -Dalanyl-n-alanine; DnO walls, walls made from cells grown in D%O position of cell fracture. The only other possible source of memmedium; HpO walls, walls made from cells grown in Hz0 medium; brane damage was the process of freezing and thawing.
Da0 membranes, membranes prepared from cells grown in DIO Often, the freeze-fractured bacteria were also exposed to tolmedium; Hz0 membranes, membranes prepared from cells grown uene before the freezing step. Prior treatment with toluene in HIO medium.
was performed to ensure that membrane fragments from the broken cells would be able to utilize exogenously added peptidoglycan substrates. It was expected that these fragments would have formed closed vesicles (11). If ihese vesicles behaved like whole cell membranes then in the absence of toluene exposure they would also have been impermeable to peptidoglycan precursors (2).
In initial experiments, membranes from freeze-fractured B. megaterium cells treated with toluene were used to synthesize peptidoglycan from the substrates UDP-[14C]GlcNAc and UDP-MurNAc-Ala-Glu-Dap-Ala-Ala.
The product was measured as chromatographically immobile material. The time course of the reaction was followed at various temperatures ( Fig. 1). All subsequent reactions were performed at 30" where the rate and extent of incorporation were greatest. As expected, the omission of UDP-Mur-NAc-Ala-Glu-Dap-Ala-Ala completely inhibited the synthesis (Fig. 1). Chromatographic analyses of the reaction mixtures in Solvent A showed that the substrate was either unchanged or was transformed to nonmigrating peptidoglycan. The product was also completely digestible by 0.25 mg per ml of lysozyme treatment for 15 min at 30" and pH 8.0. The dependence of synthesis on pH (Fig. 2) and magnesium ion concentration ( Fig. 3) was also determined.
The effect of the toluene treatment before the freeze-fracture step was examined as shown in Fig. 4. This treatment was seen to be slightly stimulatory.
To check the possibility that the peptidoglycan made zn vitro was linked by peptide cross-bridges, UDP-MurNAc-Ala-Glu- GlcNAc as the radioactive precursor in the incubation mixtures (Fig. 5). In the absence of UDP-GlcNAc, there was no synthesis of peptidoglycan but carboxypeptidase(s) did cleave the terminal 1 or 2 alanine residues from the unused substrate (Fig. 5B). When both precursors were present (Fig. 5A), immobile product was formed.
Furthermore, more alanine was released than when UDP-GlcNAc was absent. This increase in free alanine in the ,0&b-c-b-0 0 I 2 3 T I ME, HOURS  presence of both substrates was consistent with the interpretation that in addition to carboxypeptidase activity there was also transpeptidase activity which could liberate alanine while making peptide cross-links.
The experiment of Fig. 5C shows that all alanine release was blocked by penicillin as had been found previously (1). Therefore, the peptidoglycan made in the presence of penicillin was uncross-linked and retained both radioactive alanine residues at the carboxyl end of each peptide side chain. The specific radioactivity per disaccharide subunit of this material must have been greater than that of peptidoglycan made in the absence of penicillin because in this latter case radioactive alanine was released from at least some of the disaccharide subunits.
Thus, in incubations of 30 min or less where the radioactivity in alanine in peptidoglycan was the same in the presence and absence of penicillin, the polymerization of the disaccharide backbone of peptidoglycan must have been slower with penicillin present.
In summary, the peptidoglycan synthetic system using freezefractured membranes of B. megatetium 899 was very similar to that found for B. megalerium QNB1551 (1). Similar results were obtained with membrane preparations obtained by treating B. megaterium 899 cells either by sonic oscillation or by grinding with alumina. Physical Studies-One physical method used for analysis of cell wall was equilibrium density gradient centrifugation in CsCI. Walls isolated from B. megatetium cells grown in the usual H20containing medium were first treated for 5 min at 100" in Sarkosyl to remove membranes and then extracted with 1 N NaOH for 5 min at 100'. The extracted walls had a specific gravity of approximately 1.5 g per cc. In experiments where cells were grown in parallel in Hz0 and DzO, the densities of the walls grown in DzO were always greater.
However, there was some fluctuation in the densities of both Hz0 and DzO walls from experiment to experiment.
In the experiment of Fig. 6 served to reach 1.540 g per cc. Most other cell constituents, if they banded in the CsCl gradient, would have been separated from either D20 or Hz0 walls.
Proteins in general have a density of 1.3 g per cc, whereas nucleic acids have a typical density of 1.7 g per cc or greater.
Peptidoglycan synthesized by membranes prepared by freezefracturing from cells grown in Hz0 was freed from membranes by treatment with Sarkosyl followed by treatment with NaOH. The treated radioactive peptidoglycan was mixed with carrier walls isolated from growing bacteria and centrifuged in a CsCl gradient (Fig. 7A). The radioactivity banded at a density lighter than that of walls made in oiuo. This result suggested that the newly made peptidoglycan was not attached to any large pieces of previously existing wall. Very little previously formed wall should have been present in the preparation of membranes used in the incubation.
However, the radioactive peptidoglycan formed in t&o may have been attached to some cell component present in the membrane preparation other than wall. Therefore CsCl gradient analysis was performed on product from a synthetic reaction in which membranes from cells grown in Hz0 were replaced by those from cells grown in D,O (Fig. 7B). Since the synthetic reaction contained non-deuterated substrates in an He0 solution, the radioactive peptidoglycan should have had the same density in the experiments of both Fig. 7 A and B. However, if peptidoglycan synthesized in vitro were attached to some cell component fopmed before cell breakage, then the material made from D20 membranes would have had an apparent density greater than that made from Hz0 membranes.
The observed position of the radioactivity relative to the carrier walls was the same in Fig. 7, A and B. In both cases the peak positions were separated by 30% of the gradient length.
To verify that the carrier walls did not affect the sedimentation, peptidoglycan made by both kinds of membranes were also centrifuged together with carrier DzO walls (Fig. 7,C and D). In these cases the separations of the peak positions were 35% of the gradient length.
The most sensitive check of the co-sedimentation of peptidoglycan synthesized by DsO and Hz0 membranes is presented in Fig. 7E. Here radioactive material from both incubations were mixed in equal amounts.
If these substances had different densities, the width of the radioactive peak would have been broader than that of any peak where peptidoglycan from only one incubation was centrifuged (Fig. 7, A to D).
A computation can be made as to how much broader the peaks should have been. The gradients in Fig. 6 show that the displacement between the peaks of the DzO and Hz0 walls was about the same as the peak width of the D20 or Hz0 walls centrifuged separately.
As a result, the peak width of the mixture of the two types of walls was twice as great as that for either type alone. Since the gradients of Figs. 6 and 7 were centrifuged under identical conditions using the same material, the density separation between the Hz0 and DzO walls in Fig. 7 would also be equivalent to the width of either the Hz0 or DzO wall peak, a difference of approximately 10 fractions. If the newly synthesized peptidoglycan were attached to old cell material that was affected by the medium change in the same way as cell wall, the peaks of radioactive material made by DzO membranes should be about 10 fractions denser than the same material made by Hz0 membranes.
This computation is based on the fact (Fig. 9) that the density gradient is approximately linear in the region between walls and radioactive peptidoglycan. Therefore, in the gradient where the two types of radioactive peptidoglycan were mixed, the peak should have been 10 fractions or 50% broader than the profile of either type of peptidoglycan centrifuged alone. In fact, the peak width measured at the inflection point in Fig. 7E was slightly narrower than that in Fig. 7A, indicating that the peptidoglycan made by B. megaterium membranes prepared by freeze-fracturing was not covalently attached to a significant amount of previously existing cell material.
The experiments described in Figs. 8 and 9 provide additional evidence for this conclusion.
In these experiments, B. megaterium cells were grown in the presence of [W]glucose to radioactively labeled cell walls and other molecules.
In order to ensure that transient intermediates as well as stable compounds were labeled, it was essential that growth be exponential and that ture was harvested after one mass doubling (40 min) for the experiment of Fig. 9. the radioactivity not be exhausted from the medium. The increase in turbidity in Fig. 8B showed that the culture was indeed in the exponential growth phase at the time of harvest. The fact that the increase in acid-precipitable radioactivity was also linear (Fig. 8A) also showed that the cells were collected while ample [Wlglucose was still present in the medium.
Consistent with this conclusion was the finding that only 15y0 of the total radioactivity in the culture was acid-precipitable at the time of harvest.
The radioactive cells were freeze-fractured without prior toluene treatment.
Then the membrane vesicles were harvested for synthesis of peptidoglycan using UDP-[aH]GlcNAc as the radioactive substrate. The incorporation mixtures were then extracted with Sarkosyl and NaOH and centrifuged in a CsCl gradient (Fig. 9B). In this experiment the CsCl solution was adjusted to 1.380 instead of 1.383 before centrifugation in order to shift the bands toward the center of the tube. The gradient tube also contained walls isolated from the cells which were broken by the freeze-fracture procedure.
Therefore, the 14C-labeled material in the gradient was cell walls and membrane fragments from cells broken by the freeze-fracture technique.
The amount of W-labeled walls in the gradient represented the same proportion of broken cells as was used to obtain membranes for the incubation.
The UDP-[aH]GlcNAclabeled material resulted from peptidoglycan synthesis in do. The profiles in Fig. 9B showed that there was no detectable 14C radioactivity in the region of the peak of the *H-labeled peptidoglycan confirming the conclusion that the bulk of the newly made material was not linked in appreciable amounts to either old cell wall or old membrane components.
The small amount of 14C radioactivity at the top of the gradient was probably due to membrane components such as proteins solubilized by the Sarkosyl and NaOH extraction steps. This was the region where Sarkosyl and any associated lipophilic material banded in the gradient.
Also, free proteins would have been found in this density region.
There also was a small amount of *H-labeled material at this light density which could easily have been newly synthesized peptidoglycan which was not fully removed from membrane material.
A control experiment (Fig. 9A) showed that, in the absence of peptidoglycan synthesis, the profile of r4C radioactivity was the same. Two separate sets of experiments, (Figs. 7 and 9) then, have both demonstrated that the newly synthesized peptidoglycan was not linked to any substantial amounts of old wall. In the experiment of Fig. 9B, only membrane fragments would have accounted for the synthesis because any contaminating whole cells would not have been able to incorporate the externally added precursors (2). The same result was found in this experiment as ones in which the freeze-fractured cells were first treated with toluene (Fig. 7). In both sets of experiments the peptidoglycan made in oitro was much less dense than native walls. Therefore, any small contamination of membrane preparations by toluene-treated whole cells probably did not affect the density gradient results in a significant way.  Fig. 7.
Since it has already been shown that B. megatetium membranes can make cross-linked peptidoglycan (l), it seemed likely that the formation of peptide cross-bridges might have been at least partly responsible for the density characteristics of the peptidoglycan synthesized in oilro. Therefore, if penicillin were added to block cross-linking, the density profile of the synthetic product might have been changed.
The results of the experiment of Fig. 10A showed that peptidoglycan made by membranes from cells broken by grinding with alumina banded similarly to peptidoglycan made from membranes prepared by freeze-fracturing (Figs. 7 and 9). When penicillin was added to the reaction, the amount of product synthesized was diminished only by approximately 30%, but this material did not form a discrete band when centrifuged in a CsCl density gradient (Fig. 10B). The failure to band could have resulted from the peptidoglycan being heterogeneous in density and hence banding throughout the gradient.
Another explanation of the disperse profile is that the material had a small size which would have increased both the band width and time required to reach equilibrium.
In any case, the experiment does suggest that cross-linking affects the physical characteristics of the in vitro product. As predicted, in the absence of the second substrate no acid-precipitable radioactivity was found in the gradient (Fig. 10). The results in Fig. 10 were also found for incubations using membranes from cells broken by sonic oscillation and freeze-fracturing.
Before addition of the CsCl for the gradients, all samples were centrifuged to remove membranes denatured by the Sarkosyl and NaOH treatments.
This centrifugation was necessary because substrate was trapped by the denatured membranes.
If the material seen as nonmigrating product after chromatography was defined as 100% of the peptidoglycan made in those reaction mixtures where both substrates were present, then the radioactivity recovered at the various steps involved in the CsCl gradient analysis were divided typically as follows: 50% was recovered from the gradient fractions and 15% was recovered in the pellets from the preliminary centrifugations after the Sarkosyl and NaOH treatments.
Almost all of the radioactive material was of homogeneous density in the gradients of reaction mixtures to which no penicillin was added. These recovery figures were essentially identical regardless of whether penicillin was present in the synthetic reactions.
The remaining 35% of the chromatographically immobile radioactivity was unaccounted for and may have been due to the liberation of very short peptidoglycan chains from the membrane-bound enzyme by Sarkosyl and NaOH.
This small material might have been chromatographically immobile but not precipitable by trichloroacetic acid.
It is possible to estimate the size of the peptidoglycan made in vitro by sedimentation velocity centrifugation in a sucrose gradient.
The radioactive product made by membranes was freed from the synthetic enzymes by Sarkosyl and NaOH treatment.
The remaining material, which by CsCl gradient analysis (Fig. 10) was shown to be peptidoglycan of a unique density, was centrifuged through a 10 to 35% sucrose gradient.
The E. coli bacteriophage 4x174 was layered with the peptidoglycan sample to provide an internal marker for molecular weight determination.
As expected, the peptidoglycan was heterogeneous in size but approximately 35% of all the peptidoglycan sedimented more rapidly than +X174 (Fig. 11). If these two compounds had the same sedimentation characteristics, then the peptidoglycan in the heavier fraction would have had molecular weights greater than the 6 x lo6 typical of $X174 (11). By comparison, only 13% of the material made in the presence of penicillin sedimented faster than 4X174 (Fig. 11B).

4813
This decrease in the amount of rapidly sedimenting radioactivity suggested that the large size of the peptidoglycan made in the absence of penicillin was due in part to cross-linking. DISCUSSIOX In vitro synthesis of peptidoglycan from the substrates UDP-MurNAc-Ala-Glu-Dap-Ala-Ala and UDP-GlcNAc catalyzed by membrane preparations obtained from B. meguletium was demonstrated by the incorporation of radioactivity from either substrate into chromatographically immobile product. Both substrates had to be present for peptidoglycan synthesis to occur.
As expected, the reaction product formed in vi&o was degraded by lysozyme.
In experiments conducted with UDP-MurNAc-Ala-Glu-Dap-Ala-Ala labeled in the 2 terminal alanine residues, the pattern of release of alanine was consistent with previous reports (1) that B. megaterium could make cross-linked peptidoglycan.

Sedimentation
velocity experiments have established that a large portion of the peptidoglycan made in vitro by B. megatedium membranes had molecular weights greater than 6 x 106. These same experiments comparing peptidoglycan made in the presence and absence of penicillin suggested that cross-linking was at least partly responsible for the large size of the material made in the absence of the antibiotic.
The CsCl gradient profiles of the same material also indicated that transpeptidation could affect the physical properties of the biosynthetic product. Two types of density gradient experiments have demonstrated that the peptidoglycan made in vitro was not attached to significant amounts of previously formed wall or membrane material.
The difference between the density of the peptidoglycan made in vitro and that of walls isolated from growing cells could be due to the fact that free diaminopimelic acid was not added to the in vitro reaction mixtures.
Therefore, the transpeptidation dependent on free diaminopimelic acid (1) was not permitted in the in vitro product.
The resulting peptidoglycan could have had a cross-linking pattern different from that of the walls made in viva. Also, the chemical composition would certainly have been different.
In addition, any other wall polymers made by B. megaterium such as a teichuronic acid would only have been present in the walls made from growing cells because any necessary substrates were not present in the in vitro incubations.
Experiments are now in progress to study the physical properties of the peptidoglycan made in vitro in which both B. megaterium transpeptidation reactions are permitted. Substances with molecular weights in the range of IO6 to lo7 can be observed with the electron microscope. Therefore, it should be possible to examine the differences between walls made in viuo and the peptidoglycan made in oitro.
Since newly made material can be radioactively labeled with tritium, we have begun autoradiographic experiments to locate and positively identify peptidoglycan made in vitro.