The composition of the cyst wall of the beet cyst-nematode Heterodera schachtii

1. Cyst walls of the beet cyst-nematode (Heterodera schachtii Schmidt) were obtained by sieving a suspension of crushed cysts; about 15mg of dried cyst walls was obtained from 1000 cysts. 2. The cyst walls contained 68% protein calculated from nitrogen content. Glutamic acid, glycine, proline and hydroxyproline made up about 54% by weight of the amino acids obtained on acid hydrolysis. 3. Minor constituents of the cyst wall were hexosamine (3.3%), lipid (6%), carbohydrate (2%) and phenols (2%). The hexosamine was identified as galactosamine. 4. The cyst walls contained inorganic material (ash 17%), most of which was extractable with EDTA, but not with water. Major inorganic components were calcium and phosphorus (1.7% and 1.5% respectively, by weight). Carbon dioxide (about 1% by weight) was liberated from the cyst walls on acidification. 5. The cyst walls of H. schachtii and the potato cyst-nematode (Heterodera rostochiensis) contained different amounts of the same amino acids. They also differed in their inorganic content and in the nature of the hexosamine present.     

Carbohydrate and amino acid analyses of Giardia muris cysts.

Intact Giardia muris cysts were subjected to consecutive chloroform/methanol and 2% sodium dodecyl sulfate (SDS) extractions, and to amyloglucosidase treatment. The SDS-insoluble, amyloglucosidase-fast cyst walls (ACW) were further incubated with chymotrypsin, trypsin, papain, or pronase. Low voltage scanning electron microscopy revealed no discernible change in the ultrastructure of the filamentous layer of the cyst wall following any of these treatments. Affinity for cyst wall-specific monoclonal antibody (Meridian Diagnostics, Cincinnati, OH) was also retained after all treatments. Periodic acid-Schiff staining and gas chromatography/mass spectrometry (GC/MS) of intact and treated cyst hydrolysates showed a significant reduction in the amount of glucose associated with the cyst (72 nmoles/10(6) intact cysts vs 1.9 nmoles/10(6) ACW) as a result of amyloglucosidase treatment, indicating that glucose is stored within Giardia as an SDS-insoluble polymer. Galactosamine was identified by GC/MS as the predominant sugar associated with both the ACW and the proteinase treated ACW (42 nmoles/10(6) ACW). High performance liquid chromatographic analysis of amino acids from intact and treated cyst hydrolysates revealed a marked reduction, but not elimination, of detectable quantities of identifiable amino acid residues (255 nmoles/10(6) intact cysts vs 6.8 nmoles/10(6) proteinase treated ACW). These results suggest that the filamentous layer of the cyst wall is primarily a carbohydrate peptide complex.     

Carbohydrate and Amino Acid Analyses of Giardia muris Cysts

ABSTRACT. Intact Giardia muris cysts were subjected to consecutive chloroform/methanol and 2% sodium dodecyl sulfate (SDS) extractions, and to amyloglucosidase treatment. The SDS-insoluble, amyloglucosidase-fast cyst walls (ACW) were further incubated with chymotrypsin, trypsin, papain, or pronase. Low voltage scanning electron microscopy revealed no discernible change in the ultrastructure of the filamentous layer of the cyst wall following any of these treatments. Affinity for cyst wall-specific monoclonal antibody (Meridian Diagnostics, Cincinnati. OH) was also retained after all treatments. Periodic acid-Schiff staining and gas chromatography/mass spectrometry (GC/MS) of intact and treated cyst hydrolysates showed a significant reduction in the amount of glucose associated with the cyst (72 nmoles/10⁶ intact cysts vs 1.9 nmoles/10⁶ ACW) as a result of amyloglucosidase treatment, indicating that glucose is stored with in Giardia as an SDS-insoluble polymer, Galactosamine was identified by GC/MS as the predominant sugar associated with both the ACW and the proteinase treated ACW (42 nmoles/10⁶ ACW). High performance liquid chromatographic analysis of amino acids from intact and treated cyst hydrolysates revealed a marked reduction, but not elimination, of detectable quantities of identifiable amino acid residues (255 nmoles/10⁶ intact cysts vs 6.8 nmoles/10⁶ proteinase treated ACW). These results suggest that the filamentous layer of the cyst wall is primarily a carbohydrate peptide complex.     

The chemical composition of the egg shells of the potato cyst-nematode, Heterodera rostochiensis Woll

1. Eggs of the potato cyst-nematode (Heterodera rostochiensis Woll.) were isolated by sieving a suspension of crushed cysts. Eggs were broken open by ultrasonic vibration and the egg shells separated from the released larvae by centrifuging in a potassium tartrate density gradient. About 1 mg. of dried egg shells was obtained from 1000 cysts. 2. The major constituent of the egg shells was protein (59%, calculated from nitrogen content). About 80% of the egg shells went into solution on acid hydrolysis. Of the 18 amino acids determined with the Technicon Auto-Analyser, proline was most abundant and, with aspartic acid, glycine and serine, made up about 64% by weight of the total amino acids. The small amounts of aromatic and sulphur-containing amino acids, and the presence of hydroxy-proline, indicate a collagen-like protein. 3. The egg shells gave a positive van Wisselingh colour test for chitin, and glucosamine was detected in their acid hydrolysate by chromatography. The glucosamine content of the egg shells, determined by the Elson-Morgan colorimetric method, was 7%, corresponding to about 9% chitin. 4. Dried egg shells contained about 7% of lipid, 6% of carbohydrate and 3% of ash. Polyphenols (3% by weight of the egg shells) were detected in the acid hydrolysates. 5. Neither the collagen nor the chitin showed evidence of crystallinity when examined by X-ray diffraction.     

Free amino acid concentration in hydatid cyst fluids from fertile and infertile human and animal Echinococcus granulosus.

The aim of this study was to search and compare free amino acid composition of fertile and infertile cyst fluids obtained from humans and animals infected naturally with Echinococcus granulosus, by using automated analysis based on cation-exchange chromatography with post-column ninhydrin derivatization system. 11 free amino acids from fertile (sheep origin), nine from infertile (cattle origin), 13 from infertile (human origin) hydatid cyst fluids and 19 amino acids from sera of patients with hydatid infection were detected. The levels of glycine, alanine, valine and lyrosine in fertile and infertile hydatid cysts fluids were significantly higher than in sera from patients with hydatid cysts. Glycine level in the fertile hydatid cyst fluids (sheep origin) was significantly higher than those of infertile cysts fluids (cattle and human origin) and sera with hydatid patients. Glycine level in fertile hydatid cyst fluids was about two times more concentrated in infertile cattle cyst fluids, 10 times more concentrated in infertile human hydatid cyst fluids and 13 times more concentrated in sera with hydatid patients. On the other hand, alanine and valine concentration in the fertile and infertile cyst fluids were at similar level with the exception that valine level in fertile cyst fluids was 12 times more concentrated in infertile human cyst fluids. The levels of tyrosine, citrulline, leucine, isoleucine and lysine amino acids in fertile and infertile hydatid cyst fluids were similar. Our findings with respect to fertile and infertile cysts fluids showed that free amino acids concentrations in cyst fluids were significantly, higher in sera from patients with hydatid cyst. Total amount of free amino acids content in fertile and infertile cyst fluids was three to eight times higher from that of human sera with hydatid patients.     

6 Lignin-protein complex in cell walls of Pinus elliottii: Amino acid constituents

Cell walls of Pinus elliottii callus contain ca 12 % protein. Klason lignin prepared from the walls contained 9 % protein and represented 4.5 % of the wall. The lignin fraction was increased to 22 % of the wall weight by reacting washed cell-wall tissue with coniferyl alcohol and H₂O₂, a reaction catalysed by peroxidase that remained bound to the wall. The augmented lignin preparation yielded 10 % protein. The acid hydrolysate of whole wall tissue included five amino acids at a concentration higher than hydroxyproline. The hydrolysates of both natural and augmented lignin preparations yielded distributions of amino acids in which the concentration of hydroxyproline was higher than that of all other amino acids. The results suggest that polymerizing lignin links covalently with cell-wall glycoprotein, and that the bonds may be formed preferentially with hydroxyproline.     

The structure of a glycopeptide isolated from the yeast cell wall

1. Glycopeptides containing mannose were extracted from isolated yeast cell walls by ethylenediamine and purified by treatment with Pronase and fractionation on a Sephadex column. 2. A glycopeptide that appeared homogeneous on electrophoresis and ultracentrifugation had a molecular weight of 76000, and contained a high-molecular-weight mannan and approx. 4% of amino acids. 3. The amino acid composition of the peptide was determined. It was rich in serine and threonine and also contained glucosamine. No cystine and methionine were detected. 4. The glycopeptide underwent a beta-elimination reaction when treated with dilute alkali at low temperatures. The reaction resulted in the release of mannose, mannose disaccharides and possibly other low-molecular-weight mannose oligosaccharides. During the beta-elimination reaction the dehydro derivatives of serine and threonine were formed. One of the linkages between carbohydrate and amino acids in the glycopeptide is an O-mannosyl bond from mannose and mannose oligosaccharides to serine and threonine. 5. After the beta-elimination reaction the bulk of the mannose in the form of the large mannan component was still covalently linked to the peptide. This polysaccharide was therefore attached to the amino acids by a linkage different from the O-mannosyl bonds to serine and threonine that attach the low-molecular-weight sugars. 6. Mannan was prepared from the glycopeptide and from the yeast cell wall by treatment of the fractions with hot solutions of alkali. The mannan contained aspartic acid and glucosamine and some other amino acids. The aspartic acid and glucosamine were present in equimolar amounts; the aspartic acid was the only amino acid present in an amount equivalent to that of glucosamine. Thus there is the possibility of a linkage between the mannan and the peptide via glucosamine and aspartic acid. 7. Mannose 6-phosphate was shown to be part of the mannan structure. Information about the structure of the mannan and the linkage of the glucosamine was obtained by periodate oxidation studies. 8. The glucosamine present in the glycopeptide could not be released by treatment with an enzyme preparation obtained from the gut of Helix pomatia. This enzyme released glucosamine from the intact cell wall. Thus there are probably at least two polymers containing glucosamine in the cell wall. 9. The biosynthesis of the mannan polymer in the yeast cell wall is discussed with regard to the two types of carbohydrate-amino acid linkages found in the glycoprotein.     

The cyst wall of Colpoda steinii. A substance rich in glutamic acid residues

1. The cyst wall of Colpoda steinii has been isolated and its chemical nature examined. It had a nitrogen content 13.9+/-0.2% (s.d.) and an ash 8.6+/-1.6% (s.d.). After lipid and hot-acid extraction there was a variable residual phosphorus of 0.19-0.64%. The protein nature, indicated by infrared and ultraviolet absorption, was confirmed when 100mug. of hydrolysed wall gave a ninhydrin colour equivalent to that given by 0.88-1.01mumoles of glycine. Hexosamine, hexose, pentose, lipid and dipicolinic acid were absent. 2. Paper chromatography of hydrolysates, besides showing the presence of the usual protein amino acids and three unidentified ninhydrin-reacting spots, indicated the presence of large amounts of glutamic acid. Estimated by chromatography, the amount present was 52.9+/-0.6 (s.d.) g./100g. of ash-free wall; manometric estimation of l-glutamic acid with l-glutamate 1-carboxy-lyase gave 46.5+/-0.9 (s.d.) g./100g. 3. Free carboxyl groups were estimated by titration as 0.159+/-0.011 (s.d.) mole/100g. and those present as amide as 0.154+/-0.004 (s.d.) mole/100g., and the total was compared with the dicarboxylic acid content 0.360+/-0.010 (s.d.) mole/100g. 4. After treatment with 98% formic acid 25-30% of the wall material could be extracted by 0.05m-sodium carbonate solution (extract 1); after treatment of the residue with performic acid a further 62-63% based on the original weight could be extracted by 0.05m-sodium carbonate (extract 2). 5. The average values found for the glutamic acid contents were 21.7g./100g. for extract 1 and 58.0g./100g. for extract 2. The cysteic acid content of whole oxidized wall was about 5.8g./100g. and of extract 2 also about 5.8g./100g. The glutamic acid and cysteic acid contents of the final residue were also investigated. 6. The significance of these extraction experiments in relation to the wall structure is discussed.     

A chemical and autoradiographic study of cellulose synthesis during the encystment of Acanthamoeba castellanii

When cells of Acanthamoeba castellanii are placed in a non-nutrient medium, they differentiate into cysts which possess cellulosic walls. In the present study, the source of the glucosyl unit for cyst wall cellulose was investigated by following the encystment of trophozoites grown in the presence of ¹⁴C-labeled fatty acids (uniformly labeled palmitate or oleate) or [3-³H]glucose. Cells were fractionated at the beginning and after 30 hr of encystment using a modified Schmidt-Tannhauser procedure. In cells grown on fatty acids, 90% of the labeled material was in the lipid fractions both before and after encystment with the total amount of label/cell changing very little. Both partial and complete acid hydrolysis of the glycogen of the acidsoluble fraction and the alkali-insoluble residue of the cyst wall indicated that the glucose of both fractions was not radioactive, although Acanthamoeba is known to have a functional glyoxylate pathway.Fractionation data of cells grown on [³H]glucose indicated a sevenfold increase in radioactivity in the wall insoluble fraction and a fivefold decrease in the acid-soluble fraction with the cpm/cell of the other fractions changing very little after 30 hr of encystment. Approximately 70% of the ³H-labeled material was recovered as glucose from the 30-hr wall insoluble fraction following complete acid hydrolysis. The specific radioactivity of glucose in the cyst wall insoluble fraction was the same as that of glycogen glucose isolated from the acid soluble fraction of trophozoites. Electron microscopic autoradiography showed that the majority of nonlipid radioactivity was due to glycogen in trophozoites. Autoradiograms failed to reveal Golgi bodies or any particular region of the cell as being the specialized site of cellulose synthesis. The results of the fractionation and autoradiographic studies are consistent with the concept that glycogen is a precursor of cyst wall cellulose, and that glucosyl units of glycogen and/or other glucose derivatives are converted to cellulose without significant dilution under the experimental conditions used.     

Quantitative microanalysis of cell walls of Saprolegnia diclina humphrey and Tremella mesenterica fries

Cell walls of the fungi Saprolegnia diclina Humphrey and Tremella mesenterica Fries were analyzed quantitatively. Particular attention was paid to the hydrolysis and analysis of neutral sugars, amino sugars and amino acids. These components, together with total lipids, total uronic acids and the ashed residue, accounted for more than 90% by weight of the original dry cell wall preparation. There were substantial losses of amino acids during hydrolysis; however, analytical recovery approached 100% when total protein was calculated from the total nitrogen analysis. The analytical procedures were reproducible (±3% for amino acids and amino sugars, and ±5–10% for other components) when applied to individual cell wall preparations. However, even under carefully standardized conditions, different cell wall preparations from the same species showed variable composition.Glucose was the predominant neutral sugar in the cell wall polymers of both species. The amino acid compositions were remarkable in that neither species contained detectable levels of cyst(e)ine. Hydroxyproline was detected in both species. The report from Tremella mesenterica is the first for this imino acid from the cell wall of a Basidiomycete.     

Cell wall composition in the ascomycete sphaerostilbe repens at different developmental stages

Changes in the biochemical composition of isolated cell walls were analysed during the differentiation of coremia and rhizomorphs in Sphaerostilbe repens.Differentiation was accompanied by exclusively quantitative variations of the wall components: the content in carbohydrates, chitin and free amino sugars increased; on the contrary, amino acids, uronic acids, lipids and mineral substances decreased.Carbohydrates were composed of glucose, galactose and mannose; glucosamine was the main component of amino sugars. The predominant amino acid in the walls was cysteine the amount of which increased during hyphal aggregation, while quantities of the sixteen other determined amino acids decreased.Mineral matter was present in large quantities in the walls of the fungus, especially in vegetative mycelium. Iron, phosphorus and calcium were the most abundant elements.Possible relations between the variations in chemical composition of the wall and the capability of hyphae to aggregate are discussed.     

Composition of the walls of pollen grains of the seagrass Amphibolis antarctica

The composition of walls isolated from pollen grains of the seagrass Amphibolis antarctica was determined. Glucose, galactose, and rhamnose were the major neutral monosaccharides in the wall polysaccharides, and fucose, arabinose, xylose, and mannose were present in minor proportions. No apiose, a monosaccharide present in the wall polysaccharides of the vegetative parts of the seagrass Heterozostera tasmanica, was found. Large amounts of uronic acid (mainly as galacturonic acid) were found in the walls. The monosaccharides were probably present in cellulose and pectic polysaccharides, the latter comprising neutral pectic galactans, and rhamnogalacturonans containing high proportions of rhamnose. The walls contained a small amount of protein; glycine and lysine were the amino acids present in the highest proportions. Histochemical examination of isolated walls confirmed the presence of polyanionic components (pectic polysaccharides), -glucans (cellulose), and protein. The composition of the walls is discussed in relation to analyses of the walls of pollen grains and vegetative organs of other plants.     

Interrelationships between water and cellular metabolism in Artemia cysts VII. Free amino acids

The concentration of total ninhydrin-positive material (NPM) soluble in 5% trichloroacetic acid was measured in cysts of the brine shrimp, Artemia salina, as a function of hydration level. No net change in NPM was observed until the cysts had achieved a water content of about 0.65 g H₂O/g of initially dry cysts. Above this hydration threshold the NPM content increased markedly. Examination of the free amino acid composition of cysts incubated at selected hydration levels revealed that almost all of the amino acids underwent net change above the hydration threshold. However, just below this threshold, the free amino acid composition was essentially the same as in fully dried cysts. The activity generating net changes in the concentration of free amino acids above the hydration threshold was shown to be metabolic in nature and restricted to the cellular component of the cyst.     

Chemical Composition of the Cell Walls of Clostridium botulinum Type A

Cell walls were isolated by sonic disruption of log-phase cells of Clostridium botulinum type A strain 190L and purified by treatment with sodium dodecyl sulfate (SDS) followed by digestion with proteases. Electron microscopy revealed that the cell walls thus obtained were free of both cytoplasmic membrane and cytoplasmic fragments. The purified cell wall contained 8.7% total nitrogen, 15.0% total hexosamines, 22.4% reducing groups, 8.3% carbohydrate, and 3.1% glucose. The content of total phosphorus was very low (0.02%), and therefore it was expected that teichoic acid might be absent in the cell wall. The wall peptidoglycan contained glutamic acid, alanine, diaminopimelic acid, glucosamine and muramic acid in the molar ratios of 1.00:1.85:0:85:1.06:0.67. A low amount of galactosamine was also present, but no other amino acids were found in significant quantities. The SDS-treated cell walls were not attacked by lysozyme, but after extraction with hot formamide they were completely dissolved by the enzyme and released reducing groups. The lysozyme digest was separated into two constituents, the saccharide moiety and the peptide moiety on Sephadex G-50.     

Preparation and Chemical Composition of the Cell Walls of Mature Infectious Dense Forms of Meningopneumonitis Organisms

Relatively large-scale production and purification of meningopneumonitis organisms was developed for chemical and immunological studies on cell walls of the infectious dense forms. By disruption of purified organisms with glass beads in a Mickle shaker, highly purified preparations of cell walls were obtained by sucrose density gradient centrifugation, enzyme digestion, and sodium dodecyl sulfate treatment. The dry-weight recovery of purified cell walls from intact organisms was about 13%. When (32)P-labeled preparations of cell walls were fractionated into acid-soluble, lipid, ribonucleic acid (RNA), deoxyribonucleic (DNA), and residual fractions, about 80% of the (32)P in cell wall preparations was recovered in the phospholipid fraction, which corresponded to about 3% of the total phospholipid in the intact organisms. About 7% of the (32)P in purified cell walls was recovered in the RNA and DNA fractions respectively, but this corresponds to only about 0.4% of the (32)P found in those fractions in intact organisms. From dry-weight determinations, it was calculated that the purified cell wall preparations contained only 0.6% total nucleic acids, and these are probably not true cell wall constituents. These cell walls contained 70 to 75% protein, corresponding to about 14% of the protein in intact organisms. Amino acid analysis of these protein showed the existence of all common amino acids, glucosamine, and galactosamine. However, no muramic acid was detected by the methods employed.     

High-resolution electron microscopical study of cyst walls of Entamoeba spp

Knowledge of the fine structural organization, molecular composition and permeability properties of the cell surface of intestinal protozoan cysts is important to understand the biologic basis of their resistance. Recent studies on the biology of the cyst walls of Entamoeba histolytica and Entamoeba invadens have considerably advanced knowledge on the cellular processes involved in the transport and surface deposition of the main cyst wall components. Using transmission electron microscopy, cytochemistry, scanning electron microscopy and freeze-fracture techniques, we have obtained new information. In mature cysts the permeability of Entamoeba cysts is limited to small molecules not by the cyst wall, but by the plasma membrane, as demonstrated with the use of ruthenium red as an electron-dense tracer. Cell walls of E. histolytica cysts are made up of five to seven layers of unordered fibrils 7-8 nm thick. Alcian blue stains a regular mesh of fibrils approximately 4 nm thick, running perpendicularly to the cyst wall. In addition, abundant ionogenic groups are seen in cyst walls treated with cationized ferritin. In the mature cysts of E. histolytica and E. invadens small cytoplasmic vesicles with granular material were in close contact with the plasma membrane, suggesting a process of fusion and deposition of granular material to the cell wall. The plasma membrane of mature cysts is devoid of intramembrane particles when analyzed with the freeze-fracture technique. When viewed with scanning electron microscopy the surface of E. histolytica cysts clearly differs from that of Entamoeba coli and E. invadens.     

Detection of antigenic cyst wall elements in Colpoda inflata: an immunoelectron microscopic study and immunoblotting identification of cyst wall polypeptides

By using an antiserum against isolated cyst walls from resting cysts of the ciliate Colpoda inflata, cyst wall polypeptides have been identified by immunoblotting test. Likewise, an immunoelectron microscopical study on both complete resting cysts and isolated cyst walls to localize the cyst wall proteins recognized by the antiserum, has been carried out. The immunoblotting test showed that three main polypeptide bands were recognized by the antiserum, with tentative molecular weights of 61, 66 and 70 kDa respectively. This methodology provides a better identification of cyst wall proteins after electrophoretic separation of cyst wall samples from ciliate resting cysts.     

Chemical Composition of the Cell Walls of Bacillus stearothermophilus

Cell walls were isolated by mechanical disruption of mid-log phase cells of Bacillus stearothermophilus NCA 1503-4R grown in Trypticase-yeast extract-fructose medium at 55 C. The cell walls were purified by treatment with sodium dodecyl sulfate (SDS) and incubation with deoxyribonuclease and trypsin. The cell wall peptidoglycan contained glucosamine, muramic acid, alpha, epsilon-diaminopimelic acid, and glutamic acid. Low amounts of glycine, galactosamine, serine, aspartic acid, lysine, and valine were also present. The relative mole ratios of glutamic acid-alpha, epsilon-diaminopimelic acid-glycine-alanine were 1.00:1.26:0.08:1.55. The cell walls were free from ribonucleic acid and deoxyribonucleic acid and contained less than 0.2% chloroform-methanol extractable lipid and 0.09 mumole of phosphorus per mg of cell wall. Teichoic acid was not detected in the cell walls of this organism. Cell walls isolated without treatment with SDS contained 7.5% chloroform-methanol extractable lipid, 0.24 mumole of phosphorus per mg of cell wall, and relatively high concentrations of all amino acids. These results suggest that the extracted lipid is not a cell wall component per se, but a contaminant from the lipoprotein cell membrane.     

In vitro excystment of the metacercaria of Maritrema arenaria (Digenea: Microphallidae)

Metacercarial cysts of Mantrema arenaria were subjected to a solution containing trypsin and bile salts at 41°C. This treatment induced intense metacercarial activity and after 15 min metacercariae burst through their cyst walls and emerged. Electron microscopy demonstrated that during the process of excystment the inner layer of the cyst wall changed from a compact to a loose fibrous state. Experiments showed that only cysts containing viable metacercariae underwent this change whereas cysts which had been forcibly vacated before treatment did not. This indicated that the structural change of the inner layer of the cyst wall could not be attributed to the excystment medium. Also there was much less acid phosphatase activity in and on the surface of newly excysted metacercariae compared with encapsulated specimens. It was concluded that the excystment medium induced physical activity in, and the release of enzymic material by, the metacercariae. Together these activities rendered the cyst wall soft and susceptible to rupture by physical pressure.     

Studies on Cell Wall of Alkalophilic Bacillus

Cell walls of alkalophilic Bacillus No. C-125 and No. A-59 which grew in different pH conditions were prepared and analyzed. In the walls from cells grown at pH 10.3 (pH 10.3-cell wall) and the walls from cells grown at pH 7.5 (pH 7.5-cell wall) of the alkalophilic bacilli, the contents of neutral sugar and phosphorus were low as compared with those of Bacillus subtilis 6160, while uronic acid and amino acids were abundant. The uronic acid content of the pH 10.3-cell walls was higher than that of the pH 7.5-cell walls in both strains. The insoluble fraction (peptidoglycan) of cell walls of Bacillus No. C-125 consisted of muramic acid, glutamic acid, alanine, diaminopimelic acid and glucosamine as in neutrophilic bacilli. In the TCA soluble fraction of pH 10.3-cell walls of Bacillus No. C-125, uronic acid was a polymer of glucuronic acid containing a small amount of hexosamine, and 2/3 of the ninhydrin positive material was glutamic acid which was derived mainly from poly γ-L-glutamic acid.