1A0 I
CHEMICAL COMPOSITION OF THE PEPTIDOGLYCAN
OF VITREOSCILLA STERCORARIA
THESIS
Presented to the Graduate Council of the
North Texas State University in Partial
Fulfillment of the Requirements
For the Degree of
MASTER OF SCIENCE
By
Gary Levit, B. A.
Denton, Texas
August, 1976 Copyright by
Gary Levit
1976 To Dr. A. S. Kester ABSTRACT
Levit, Gary, Chemical Composition of the Peptidoglycan of Vitreoscilla stercoraria. Master of Science (Biology) ,
August, 1976, 30 pp., 2 tables, bibliography, 25 titles.
The peptidoglycan layer of Vitreoscilla stercoraria,
ATCC 15218, was isolated from intact cells after treatment with sodium lauryl sulfate (SLS) and digestion with Pronase.
Amino acid and amino sugar content was analyzed and
67% of the total present was made up of glutamic acid, ala nine, diaminopimelic acid (DAP), and glucosamine in a molar ratio of 1:1.7:1:0.7.
Electron microscopy of the final peptidoglycan product showed a thin, delicately folded sacculus which exhibited a morphology different from that of the intact vegetative cells.
Within these sacculi occurred electron-dense structures which were assayed and found to be poly- 3-hydroxybutyrate (PHB) granules.
The final yield of peptidoglycan was 2.9% of the dry weight of the intact vegetative cell. TABLE OF CONTENTS
Page
LIST OF TABLES - - W -. iv
LIST OF ILLUSTRATIONS.
INTRODUCTION.S-....
MATERIALS AND METHODS* 7
RESULTS...... -.....-.. .13
DISCUSSION....-.-.-.-.. . 025 LITERATURE CITED - .29
iii LIST OF TABLES
Table Page
I. Rf Values for Diaminopimelic Acid (DAP) Isomers and Peptidoglycan Hydrolysate for Vitreoscilla stercoraria...... 22
II. Amino Acid and Amino Sugar Analysis of Pep todoglycan of Vitreoscilla stercoraria . . . 23
iv LIST OF ILLUSTRATIONS
Figure Page
1. Basic Structure of the Peptidoglycan of a Gram-negative Organism...... 2
2. Purification of Peptidoglycan of Vitreo scilla stercoraria...... 9
3. Growth Curve for Vitreoscilla stercoraria in Tryptic Soy Broth (TSB)...... 14
4. Electron-micrograph of Vitreoscilla stercoraria on Formvar-coated Grid (XIOOOO)...... 15
5. Photomicrograph of the Gram Reaction of Vitreoscilla stercoraria (X1,000).w...... 16
6. Photomicrograph of a Sudan Black B stained Preparation of Vitreoscilla stercoraria (Xl,000)...... 17
7. Electron-micrograph of pellet after SLS treatment of Vitreoscilla stercoraria (X15,500)...... 19
8. Electron-micrograph of Pellet after Pronase Treatment of Vitreoscilla stercoraria (X15,500)...... 20
9. Electron-micrograph of Final Peptidoglycan Purification from cells of Vitreoscilla stercoraria...... 21
10. Proposed Chemical Structure of the Peptido glycan layer of Vitreoscilla stercoraria. . 26
V INTRODUCTION
There are many unique features in the structure of the bacterial cell wall which are of great interest and impor
tance. For instance, the cell wall (a) contains surface
antigens which interact with the immune system of a suitable host, (b) contains structures and chemical compounds which can be used in taxonomic differentiation of bacteria,
(c) appears to have a direct influence on the staining char acteristics of an individual cell, (d) exhibits a high degree of specificity in bacteriophage adsorption, and (e) is the point of attack of some antibiotics. Contained within the cell wall is an electron-dense structural layer which is thought to be responsible for giving each bacterial cell its characteristic morphology. This rigid, covalently linked framework surrounds the bacterial cell and is actually one large sac-like molecule called the peptidoglycan layer (6).
The essential features of this sacculus, found in Gram-nega tive bacteria, are a backbone of alternating N-acetylgluco samine and N-acetylmuramic acid residues in 3-1,4 linkage, a tetrapeptide side chain with alternating L and D amino acid residues, and a peptide bridge from the terminal car boxyl of one tetrapeptide to an available NH 2 -group of a neighboring tetrapeptide (Fig. 1). Included in the tetra peptide side-chain are L-alanine, D-alanine, D-glutamate,
1 2
Y Y Ala Ala D9In D-GIn X-PX.m-AP D-A D-Ala
Fig. 1. Basic structure of the peptidoglycan of a Gram-negative organism. The backbones are made up of X which is an N-acetylglucosamine residue, and Y an N-acetylmuramic acid residue. The tetra peptide side-chains of each disaccharide unit are cross-linked to those of adjacent chains by a peptide bridge. 3 and either diaminopimelic acid or its decarboxylation product
L-lysine. Within this framework, many variations are found; muramic acid may occur as N-glycolymuramic acid, the amino acids in the peptide bridge vary from organism to organism, and the degree of cross-linking varies in frequency (3, 12).
Although other wall layers contribute to rigidity in some way, it has been shown that cells bounded only by the pep tidoglycan layer maintain their shape (14).
Electron microscopy of a Gram-positive cell shows a
0 thick (200-800 A), dense outer layer which surrounds a thin ner (75 A) plasma membrane (3). The plasma membrane has a protein content of 60 to 70% with 20 to 30% lipid and small amounts of carbohydrate. The thick layer is the peptidoglycan and constitutes 30 to 70% of the total wall. It may have covalently attached to it polypeptides, polysaccharides, or teichoic acids. The latter two are believed to have antigenic significance (8). Electron microscopy of the Gram-negative cell wall shows three major layers. There is an outer membrane (60 to 180 A) which is composed mainly of phos pholipids, lipopolysaccharides, and proteins (2). Beneath this membrane lies what is called the "periplasmic space"
(13). Martin describes this "space" as an enzyme-containing compartment bounded on the inside by the cytoplasmic membrane and on the outside by a "molecular sieve." Within the peri plasm is an electron-dense layer (20 to 30 A) which repre
sents less than 10% of the Gram-negative cell wall. 4
This dense layer corresponds to the thick peptidoglycan of the Gram-positive bacteria. The cytoplasmic membrane lies innermost in the wall and contains approximately 30% lipid,
60% protein, and 2% carbohydrate (13). The peptidoglycan is recognized as the only cell wall polymer common to both
Gram-positive and Gram-negative bacteria.
Although the walls of Gram-positive bacteria reveal a great variation in the composition and structural arrangement of their peptidoglycans, all Gram-negative organisms pre viously studied for amino acid composition have shown only slight variation. The tetrapeptide ha-s been shown to contain the amino acids D-glutamic acid, meso- diaminopimelic acid, and alanine (D and L) in a molar ratio of 1:1:2 (20). Much interest has been shown in the peptidoglycan layer and work has been carried out in this area to elucidate its biosyn thesis and chemical structure as it occurs in different organisms (7, 8, 14, 171 18, 21, 22, 23, 24).
It is the purpose of this work to determine the chemical composition of the peptidoglycan layer of the gliding bac
terium Vitreoscilla stercoraria. According to the Eighth
Edition of Bergey's Manual of Determinative Bacteriology (1),
the genus Vitreoscilla belongs in the family Beggiatoacae which is located in Part 2, "The Gliding Bacteria." These organisms grow as filaments containing cells in chains. The
filaments are flexible, motile by gliding, Gram-negative, and
commonly exhibit poly- -hydroxybutyrate granules within 5 the cell. The genera of this family are distinguished by the presence or absence of sulfur granules deposited when cells are grown in the presence of H2 S and/or by the ability or lack of ability of the organism to form a well-defined sheath. Vitreoscilla stercoraria is a non-sheathed organism which does not deposit sulfur granules. It is further stated in Bergey's Manual that although the genera are apparently recognizable, this grouping may be precarious inasmuch as their affinities are uncertain because of the lack of criti cal information about these organisms.
The only published information to date on the peptido glycan layer of the gliding bacteria has been that of Verma and Martin,who studied the genera Cytophaga and Sporocytophaga,
(25) and Dworkinconcerning the genus Myxococcus xanthus (4).
Verma and Martin found that peptidoglycan was a major cell component in enzyme-treated vegetative cells as well as in the microcysts of these non-fruiting myxobacteria. The amino sugar and amino acid constituents of the peptidoglycan of myxobacteria were found to be muramic acid, glucosamine,
2,6-diaminopimelic acid (DAP), glutamic acid, and alanine in molar ratios of 1:1:1:1.2. In similar experiments, Dworkin
(4) found that both vegetative cells and microcysts of M. xanthus contain approximately 0.6% peptidoglycan (by weight).
The overall composition of the peptidoglycan is similar in both cell types, containing muramic acid, glucosamine,
DAP, glutamic acid, and alanine in a molar ratio of 6
0.75:0.75:1:1:1.7. Dworkin also found that treatment of
M. xanthus vegetative cells with trypsin and sodium lauryl
sulfate caused complete disaggregation of the walls, sug
gesting a discontinuous peptidoglycan layer.
The value of a study of the peptidoglycan layer of V.
stercoraria was justified by the limited knowledge of the peptidoglycan of the gliding bacteria and the paucity of
information concerning the genera of the family Beggiatoacae. MATERIALS AND METHODS
Organism. Vitreoscilla stercoraria ATCC 15218, was maintained on Trypticase Soy Agar slants (Difco).
Growth conditions. Large numbers of cells were obtained by the inoculation of 50 ml of Trypticase Soy Broth (TSB)
(Difco) in 250-ml Erlenmeyer flasks with 5.0 ml of a sus pension of cells washed from a 48-hour-old agar slant culture.
The culture was incubated at 25-28 C on an shaker (150 rev/min) until it reached the log phase of growth (approximately 48 hr). The 55-ml broth culture was then transferred into a fermenter (Fermentation Design Inc.) containing 3 L of TSB.
These cultures were then incubated at 25-28 C with 200 rpm agitation and 1.5 1/min filter-sterilized air. When the culture reached the late log phase of growth, the cells were harvested and centrifuged in Sorvall 315-ml stainless steel centrifuge bottles at 8,000 rpm for 5 min,using a Sorvall
SS-3 centrifuge. This process yielded approximately 250 g of cells (wet weight) with an average yield of 8.3 g cells (wet weight) per liter of medium. The brownish-white paste was frozen at -20 C until used.
Purification of peptidoglycan. The peptidoglycan of V. stercoraria was prepared from intact cells by the treatment
7 8 originally described by Kolenbrander and Ensign (10). A
flow diagram of this procedure is presented in Fig. 2.
Frozen cell paste (50.0 g wet weight) was suspended in 96 ml water by mixing with a magnetic stirrer at 5 C. A 0.2-ml
aliquot was removed and the dry weight was determined. The
suspension was made 1.0% with respect to sodium lauryl sul
fate (SLS) by dropwise addition of 4.0 ml of a 25% stock
solution. The temperature of the suspension was raised to
25 C, and 1.0 mg of deoxyribonuclease (Worthington Biochemi cal Co.) was added to reduce viscosity. The mixture was then
stirred for 12 hr at room temperature.
The SLS-treated cells were centrifuged at 1,000 X g for 10 min and then at 13,000 X g for an additional 10 min at 4 C. A white layer covered a brown pellet of debris.
The dark brown supernatant was discarded. The white layer was washed from the pellet with distilled water, suspended in 200 ml water, and immediately placed in a boiling water bath for 60 min to inactivate autolytic enzymes. The heated suspension was centrifuged as above. Again a small brown pellet appeared covered by a white layer. The white top layer was washed from the pellet, resuspended in distilled waterand recentrifuged as before. The pellet was again suspended in 96 ml water and made 1.0% with respect to
SLS by dropwise addition of 4 ml of a 25% stock solution.
The suspension was mixed for 12 hr with a magnetic stirrer at room temperature and centrifuged as before. The, pellet 9
Fig. 2. Purification of Peptidoglycan of Vitreoscilla stercoraria Whole Cell Suspension.
1% SODIUM LAURYL SULFATE(SLS) FINAL SUPERNATANT (SUP) CONCENTRATION; 0.2 MG OF DEOXYRIBO (DISCARD) NUCLEASE; MIX 12H, ROOM TEMPERATURE; 1,000 X g, 10 MIN, 4 C, 13,000 X G, 10 MIN, 4 C. PELLET RESUSPENDED IN WATER; BOILING WATER BATH, SUP 60 MIN; 1,000 X G, 10 MIN, 4 C, 13,000 X G, (DISCARD) 10 MIN, 4 C. PELLET RESUSPENDED IN WATER; 1% SLS FINAL CON CENTRATION; MIX 12H, ROOM TEMPERATURE; SUP 1,000 X G, 10 MIN, 4 C, 13,000 X G, (DISCARD) 10 MIN, 4 C. PELLET SUP RESUSPENDED IN WATER; 12,000 X G, 10 MIN, (DISCARD) 4 C, REPEAT 16 TIMES. PELLET SUP RESUSPENDED IN WATER; DIGESTION WITH (DISCARD) PRONASE, 4H, 65 C, BOILING WATER BATH, 20 MIN; 12,000 X G, 30 MIN, 4 C. PELLET SUP 5.0 X 10~ 4 M EDTA; 13,000 X G, 10 MIN, 4 C, (DISCARD) REPEAT 6 TIMES, THEN 13,000 X G, 50 MIN, 4 C, WASH IN WATER, 13,000 X G, 10 MIN, C, REPEAT 10 TIMES. PEPTIDOGLYCAN 10 obtained was suspended in water and centrifuged at 12,000
X g for 20 min. This washing procedure was repeated 16
times. The final cream-colored residue was suspended in 75 ml of a 0.025 M tris (hydroxymethyl) aminomethane (Tris) buffer (pH 7.85). The suspension was heated to 65 C, and enough CaCl2 '2H2 0 added to give a final concentration of
10-4 M. Five ml of a distilled water solution containing
1.0 g of Pronase (grade B, Calbiochem, Los Angeles, Calif.) was activated by incubation in an 80-C water bath for 5 min and added to the suspension. The suspension was placed on a rotary shaker at 200 rpm at 65 C for 4 hr. The solution was then immediately placed in a boiling water bath for 20 min to destroy residual Pronase. The suspension was then centrifuged at 12,000 X g for 30 min. The pellet was washed six times by suspending it in a 5.0 X 10~4 M sodium ethyl enediaminetetracetic acid (EDTA) solution, followed by cen trifugation at 13,000 X g for 50 min. This was followed by
10 distilled water washes. The resulting white peptidoglycan paste was lyophilized, using a lyophilizer, and was stored at room temperature.
Electron microscopy. A 0.1-ml sample was removed from the suspension of original log phase cells as well as from the first and second SLS treatments, from the Pronase digest, and from the final peptidoglycan product. All samples were diluted 1:10 in water and one drop of each was placed on 11
collodion-coated grids,using a 1 cc Tuberculin syringe with
23 -gauge needle. The grids were dried in vacuo within a
glass desiccator for 1 hour and were then stained with uranyl
acetate (2%) for 5 sec. The grids were examined by means of an RCA EMU-3G electron microscope.
Analytical procedures. Samples of peptidoglycan containing approximately 100 nM of amino acids and amino
sugars were hydrolyzed in 6 N HCI at 110 C in sealed tubes overnight. The hydrolysates were evaporated to dryness and then dissolved in 2.0 ml of 0.2 N citrate buffer (pH 2.2) and analyzed on a Beckman model 120-C amino acid analyzer
(Beckman Instruments Inc., Fullerton, Calif.)
Diaminopimelic acid configuration. Two-dimensional chromatography was performed on the purified peptidoglycan to determine the isomeric form of diaminopimelic acid (DAP).
A 10-mg sample of acid-hydrolyzed peptidoglycan was applied to Whatman no. 1 filter paper and developed by two-dimensional chromatography using the solvent system of methanol., water,
10 N HCI, and pyridine in the ratio of 80:11.5:2.5:10 (19).
Thin-layer chromatography was also employed. A 5.0-mg sample of acid-hydrolyzed peptidoglycan was applied1 using the sol vent system described above. A standard mixture of meso- and
L L-DAP was co-chromatographed in each experiment.
Determination of polybetahydroxybutyric acid.
Polybetahydroxybutyric acid (PHB) was assayed by the method 12 of Law and Slepecky (11). A 10-mgm sample of the purified peptidoglycan was boiled 2 min in CHCI 3 . The residual CHC1 3 was evaporated and 10 ml of H2 SO4 were added. The solution was heated 10 min at 100 C, cooledand its light absorption at 235 nm determined with a Spectronic 210 UV spectropho tometer (Bausch & Lomb, Rochester, N. Y.). RESULTS
Vitreoscilla stercoraria cultures grew rapidly in TSB at room temperature. The growth curves (Fig. 3) of cul tures in fermenters showed the log phase of growth to end abruptly at approximately 48 hrs. To produce the large amount of cells needed for the peptidoglycan purification procedure, all fermenter cultures were harvested at 48 hr.
Mayfield (16) found in physiological studies of V. stercoraria that growth was directly proportional to tri chome length. Thus, log phase cells exhibited gradually lengthening chains until the characteristic filamentous morphology was reached (Fig. 4, 5, 6). These chains were very stable and retained their integrity even after several minutes of vigorous mixing on a Vortex mixer. Cultures of
V. stercoraria were invariably Gram-negative (Fig. 5). A large number of dense granules was observed on slides of cells stained with Sudan Black B (Fig. 6).
The procedure used in the isolation of V. stercoraria peptidoglycan (Fig. 2) made it relatively easy to obtain this layer free of the cytoplasmic membrane and of other cell wall components. The first SLS treatment initiated the solubilization of the lipopolysaccharide (LPS) containing outer membrane and of the lipoprotein of both the outer and cytoplasmic membrane, causing cell leakage.
13 RunI * 14 Run 2 E
9 8
71 m 6
5 * * * * * * **********
OD
31
a*
21 02 0 4 5060 0 8 T*E(r
1 I Fi.Grwh . urefo itescll tecrai in ryti Sy roh TS)* 15
0 * e
Fig. 4. Electron-micrograph of Vitreoscilla stercoraria on a formvar-coated grid (x 10,000). Note characteristic barrel-shaped morphology. Marker represents 1.0 p. 16
Fig. 5. Photomicrograph of the Gram reaction of Vitreoscilla stercoraria (x 3,000). 17
1 40V & l
I SOW
. to .4
Fig. 6. Photomicrograph of a Sudan Black B stained preparation of Vitreoscilla stercoraria (x 3,000). Note the dark granules within the cells. 18
Phase contrast microscopy showed minor fragmentation of the trichomes but, in general, a majority retained their original length and cell leakage was minimal. However, electron microscopy (Fig. 7) of the pellet after the second
SLS treatment showed that the trichomes were completely separated into single units. Extensive cell debris is evidenced by the dark-staining amorphous material outside the cells. The transparent peptidoglycan sacculus appeared after the Pronase treatment (Fig. 8). This micrograph, which was made prior to extensive washings, shows that much of the proteinaceous debris had been removed. Electron microscopy of the uranyl acetate-stained final peptidoglycan showed a thin, delicately folded, transparent sacculus (Fig.
9). Electron-dense granules were observed inside the cell wall sacculi. Upon staining with Sudan Black B they were
found to contain lipid. Upon applying the assay of Law and
Slepecky (11), the electron-dense structures were found to be granules of poly--hydroxybutyrate (PHB). In fact, 48 per cent of the dry weight of the final purified powder was
found to be PHB. The association of PHB with the peptido glycan has been reported previously (15).
Chromatographic analysis revealed the DAP present in the peptidoglycan to be in the meso-configuration (Table 1).
Amino acid analysis of the final, washed residue showed a heterogeneous mixture (Table 2). Only 13 of the 20 amino 19
.4
.~ ~
40
9 I Nh * 44
'9
4P ~ I
-v #.46 q
I... I. ita
'IF.
A.. * I '9*
'9
A;
Fig. 7. Electron-micrograph of pellet after SLS treatment of Vitreoscilla stercoraria (x 15,500). Marker represents 1.0 v. 20
4 & 4 ) 4
* . A 4 *,
N -4...
P
q
* * ( - . 6 * '4"
. A
Fig. 8. Electron-micrograph of pellet after Pronase treatment of Vitreoscilla stercoraria (x 15,500). Note appearance of sacculus. Marker represents 1.0 11. 21
Fig. 9. Electron-micrograph of final peptido glycan purified from cells of Vitreoscilla stercoraria (x 23,000). Note the granules of poly- -hydroxybutyrate. Marker represents 1.0 p. 22
Table 1. Rf Values for Diamonopimelic Acid (DAP) Isomers and Peptidoglycan Hydrolysate for Vitreoscilla stercoraria.
Solvent Compound12
L,L-DAP 0.43 cm 0.20 cm meso-DAP 0.82 cm 0.33 cm
Hydrolysate 0.87 cm 0.33 cm
Solvent: 1. Butanol:HOAc:H 2 0 (60:30:10 v/v) 2. MeOH:H 2 0:10 N HCl:Pyridine (80:11:2.5:10 v/v) 23
Table 2. Amino Acid and Amino Sugar Analysis of Peptidoglycan of Vitreoscilla stercoraria.
Components nM values/0.25 mg mg yield
Diaminopimelic acid 92.91 0.0177 Alanine 178.26 0.0159 Glutamic acid 106.77 0.0157 Glucosamine 77.5 0.0130 Leucine 39.43 0.005 Lysine 28.73 0.0042 Isoleucine 33.98 0.004 Phenylalanine 25.87 0.004 Glycine 46.34 0.003 Valine 22.09 0.0026 Aspartic acid 18.37 0.0024 Histidine 13.77 0.002 Threonine 12.99 0.0015 Serine 12.26 0.0012 Tyrosine 4.01 0.001 24
acids were present in a measurable amount. Of the total
amino acids and amino sugars present, 67% were made up of
glutamic acid, alanine, diaminopimelic acid (DAP), and glu
cosamine in a molar ratio of 1:1.7:1:0.7. The presence of
high levels of DAP, peculiar to the peptidoglycan of bac
teria, supports the microscopic evidence that the material
analyzed was in fact the peptidoglycan layer. The presence
of the other amino acids is believed to represent contamina
ting protein which was not removed by the various treat ments and washings. The final yield of peptodoglycan was
2.9% of the dry weight of the intact vegetative cell. DISCUSSION
Many procedures have been described for cell dis
ruption. The first method employed in this study involved
fractionation of cells disrupted with glass beads and
vigorous agitation. This method proved to be too harsh,
and purified peptidoglycan could not be obtained. The
alternate procedure involved the use of whole cells as a
starting material (10). This procedure has been shown to
be valid with intact Spirillum serpens through final product
analyses (10). The four major amino acids found were those
commonly associated with the peptidoglycan and were present
in ratios similar to those reported for the other gliding bacteria (4, 25).
The results of this experiment are summarized in a model for the peptidoglycan layer of V. stercoraria cell walls (Fig. 10). Although the N-acetylmuramic acid content was not determined, this compound is commonly described as being a component of the "backbone" molecule (3). Further more, since studies to determine the type and extent of cross-linkage occurring between the peptidoglycan tetra peptides were not undertaken, the figure is a one-dimensional representation of the carbohydrate "backbone" and its peptide moiety only. It might be mentioned, however, that a direct cross-linkage between DAP and D-alanine has been
25 26
Fig. 10. Proposed chemical structure of the peptidoglycan layer of Vitreoscilla stercoraria.
N-Acetylglucosamine N-Acetylmuramic Acid
CH2OH CH2 OH
OH NH0 H NHAc 1HAd 0 H3C-2H-$ O IjH H-CH3 L-Ala H -C
H -COOH D-Glu 9H2 H2 =O NH HC-(CH2)3-gH-COOH m-DAP. 'O H2 1YH HC-CH3 D-AlaCOOH 27
found in the Gram-negative species, Escherchia coli, Proteus mirablis, and Aerobacter cloacae (10). Moreover, the tetra peptides of all Gram-negative cell walls are thought to be
directly cross-linked (20). On the other hand, in this
study, glycine was found in a relatively high quantity
(half that of DAP) (Table 1), suggesting that it might be part of the cross-bridges of the tetrapeptides, although, to date, it has not been reported in this molecular locus among Gram-negative bacteria.
The progression of cellular changes appearing in the electron micrographs (Fig. 7, 8, 9) due to the detergent and enzymatic treatments were accompanied by a notable change in basic morphology. The normal vegetative cells of
V. stercoraria appear "barrel-shaped" (4, 5, 6). As lipo proteins and lipopolysaccharides were removed, the mor phology became gradually ovoid or spherical. The fact that the morphology of the final purified peptidoglycan is different from that of the vegative cell would seem to suggest that, in this organism, the peptidoglycan is not totally responsible for maintaining cell shape. Henning (9) states that there is no question that peptidoglycan has shape-maintaining properties. In rod-shaped organisms, in hibition of peptidoglycan synthesis or its degradation by lysozyme leads to the formation of spheroplasts. It should be pointed out, however, that it is not clear whether 28 peptidoglycan constitutes the only structure that maintains a cell shape. That the peptidoglycan is not the only structure determining cell shape is suggested by Dworkin's
(4) findings that the peptidoglycan is discontinuous in the vegetative cells of Myxococcus xanthus. Although the morphological changes induced in V. stercoraria would not seem to indicate a discontinuous peptidoglycan, they suggest that the tetrapeptides may not have extensive cross linkage, or that, in fact, the peptidoglycan layer is not totally responsible for cell shape. LITERATURE CITED
1. Bergey's Manual of Determinative Bacteriology. 8th Edition. The Williams and Wilkins Co., Baltimore, Md.
2. Costerton, J. W. 1970. The structure and function of the cell envelope of Gram-negative bacteria. Rev. Can. Biol. 29:299-316.
3. Davis, Bernard D. 1973. Microbiology. Harper and Row, Publishers, Inc. Maryland.
4. Dworkin, M., and D. Tipper. 1968. Peptidoglycan of Myxococcus xanthus: Structure and relation to morph ogenesis. J. Bacteriol. 95(6):2186-97.
5. Forsberg, C. W. 1970. Separation and localization of the cell wall layers of a Gram-negative bacterium. J. Bacteriol. 104:1354-1368.
6. Forsberg, C. W. 1972. Isolation, characterization, and ultrastructure of the peptidoglycan layer of a marine Pseudomonad. J. Bacteriol. 109:895-905.
7. Ghuysen, J. M. 1968. Use of bacteriolytic enzymes in determination of wall structure and their role in cell metabolism. Bacteriol. Rev. 32:425-464.
8. Ghuysen, J. M., J. L. Strominger, and D. J. Tipper. 1968. Bacterial cell walls, p. 53-104. In M. Florkin and E. H. Stotz (ed.) Comprehensive Biochemistry, Vol. 26A. American Elsevier Publishing Co., New York.
9. Henning, U., U. Schwarz. 1973. Determinants of cell shape. pp. 413-438. In Loretta Leive. (ed.) Bacterial membranes and walls. Marcel Dekker, Inc., New York.
10. Kolenbrander, P. E., J. C. Ensign. 1967. Isolation and chemical structure of the peptidoglycan of Spirillum serpens cell walls. J. Bacteriol. 95:201-210.
11. Law, J. H., R. A. Slepecky. 1961. Assay of poly- hydroxybutyrate. J. Bacteriol. 82:33-36.
12. Lehninger, A. L. 1970. Biochemistry. Worth Publishers, Inc. New York.
29 30
13. Martin, E. L. 1971. Isolation and chemical composition of the cytoplasmic membrane of a Gram-negative bacteria. J. Bacteriol. 105:1160-1167.
14. Martin, H. H. 1966. Biochemistry of bacterial cell walls. Annu. Rev. Biochem. 35:457-483.
15. Martin, R. J. 1963. On the nature of the granules of the genus Spirillum. Arch. Mikrobiol. 44:334-343.
16. Mayfield, D. C., A. S. Kester. 1972. Physiological studies of Vitreoscilla stercoraria. J. Bacteriol. 112:1052-1056.
17. Osburn, M. J. 1969. Structure and biosynthesis of bacterial cell walls. Annu. Rev. Biochem. 38:501-538.
18. Perkins, H. R. 1963. A polymer containing glucose and aminohexuronic acid isolated from the cell walls of Mirococcus lysodeikticus. Biochem. J. 86:475-483.
19. Rhuland, R. E., E. Work. 1955. The behavior of the isomers of DAP on paper chromatographs. J. Amer. Chem. Soc. 77:4844-4846.
20. Schleifer, K. H. 1972. Peptidoglycan types of bac terial cell walls and their taxonomic significance.
21. Schleifer, K. H. 1969. Die Murein typen bei gram positive Bakterien. Zentrabl. Bakteriol. Parasitenk, Infektionskr. Hyg. Abt. 1. Orig. 212:443-451.
22. Sharon, N. 1969. The bacterial cell wall. Sci. Amer. 220:92-98.
23. Tinelli, R. 1968. Le glycopeptide des parois bacter iennes. Bull. Inst. Pasteur. 66:2507-2538.
24. Tipper, :D. J. 1970. Structure and function of peptido glycans. Int. J. Syst. Bacteriol. 20:361-377.
25. Verma, J. P., H. H. Martin. 1967. Surface-structure of myxobacteria. I. Chemistry and morphology of the cell walls of Cytophaga hutchinsonii and Sporocyto phaga myxococcoides. Arch. Mikrobiol. 59:355-380.