Phospholipid Composition of Bacillus subtilis

Transcription

1 JOURNAL OF BACTERIOLOGY, July 1969, p Copyright i 1969 American Society for Microbiology Vol. 99, No. 1 Printed in U.S.A. Phospholipid Composition of Bacillus subtilis J. A. F. OP DEN KAMP, I. REDAI,' AND L. L. M. VAN DEENEN Laboratory of Biochemistry, University of Utrecht, Vondellaan 26, Utrecht, The Netherlands Received for publication 24 April 1969 Bacillus subtilis contained at least five phospholipids, four of which have been isolated and identified as a polyglycerol phospholipid, probably cardiolipin, phosphatidylglycerol, phosphatidylethanolamine, and lysylphosphatidylglycerol. Further purification of the latter phosphoglyceride was obtained by high-voltage electrophoresis, and it was shown that this treatment removed amino acid-containing, nonlipidic material from the phosphoglyceride. This associated material, which is not covalently linked to the lipid, gave rise to minor amounts of a number of amino acids, other than lysine, in acid hydrolysates of the lysylphosphatidylglycerol. The phospholipid composition of B. subtilis appeared to depend on the growth conditions. Addition of glucose to the medium lowered the ph during growth; this was accompanied by an increase in the amount of lysylphosphatidylglycerol and a decrease in the phosphatidylglycerol content, when compared with growth at neutral ph. The amount of the other phospholipids and the total amount of phospholipid remained constant under the different conditions. The shape and the osmotic susceptibility of the protoplasts of this organism appeared to depend on the growth conditions. Cells harvested from a neutral growth medium gave spherical protoplasts which lysed rapidly, whereas cells grown in an acidic medium maintained their rod-shaped form to a great extent after the cell wall had been removed, even after being suspended in a hypotonic medium. The latter observation suggests the presence of a more rigid membranous structure in cells which have been exposed to a low environmental ph during growth. In certain gram-positive bacteria, the phospholipid composition depends on the growth conditions, especially on the acidity of the medium during growth (9). Op den Kamp et al. (12) reported that the phosphatidylglycerol content of Bacillus megaterium, although high in cells grown at ph 7.2, is decreased considerably in organisms harvested from an acidified medium. Staphylococcus aureus and Streptococcus faecalis react in a similar way when exposed to a low ph. The decrease in the amount of phosphatidylglycerol in these organisms is accompanied by an accumulation of Zwitter-ionic phospholipids: lysylphosphatidylglycerol accumulates in S. aureus (8) and S.faecalis (9) and glucosaminyl phosphatidylglycerol accumulates in B. megaterium (13). Cultivation of B. megaterium at low ph also influences the morphology and osmotic properties of the protoplasts derived from this organism. It has been suggested that the environmental conditions may influence the chemical composition and physicochemical properties of the bacterial membranes, resulting, in the case of B. megaterium, in substantial variations in the I Present address: Department of Microbiology, University of Debrecen, Debrecen, Hungary. morphological features of the protoplasts (13). A similar relationship between environmental conditions, phospholipid composition, and properties of the protoplasts has been found in Bacillus subtilis. This paper deals with the phospholipid composition and some properties of the protoplasts of cells of B. subtilis grown under different conditions. MATERIALS AND METHODS Growth of bacteria. B. Subtilis (strain Marburg) was cultivated at 37 C in 1-liter portions under strong aeration in two different media. Medium A contained 10 g of peptone (Difco), 10 g of yeast extract (Difco), 5 g of sodium chloride, and 400 mg of sodium phosphate per liter of water (ph 7.0). Medium B contained, in addition, 20 g of glucose and 2 g of ammonium sulfate per liter. Labeling of the phospholipids was obtained by the addition of 300 sic of 32P-orthophosphate per liter of medium. The cells were harvested after growth overnight by centrifugation and were washed with distilled water which was acidified to ph 5.0 Lipid composition. The cells were lyophilized, weighed, and extracted with chloroform-methanol buffer mixtures by the procedure of Bligh and Dyer (3). Lipid extracts were dried in vacuo, weighed, and analyzed for their phosphorus content (7) to deter- 298

2 VOL. 99, 1969 PHOSPHOLIPID COMPOSITION OF B. SUBTILIS 299 mine the amount of phospholipid per gram (dry weight) of bacteria. The phospholipids were separated on silica-impregnated paper by use of the solvent system of Marinetti et al. (11). The relative amount of each phospholipid was measured by scanning the chromatograms in front of an end-window, Geiger- Muller tube. Separation of the phospholipids. Lipid extracts were fractionated on silica columns (Mallinckrodt Chemical Works; 100 to 200 mesh) with a discontinuous gradient of methanol in chloroform. Final purification of the different phospholipids was achieved by preparative thin-layer chromatography on Silica Gel G (Merck) with chloroform-methanol-acetic acid-water (250:74:19:3, v/v). Identification of the phospholipids. Preliminary identification of the phospholipids was obtained by chromatographic comparison with known phospholipids on silica-impregnated paper in diisobutyl-ketone acetic acid-water (40:25:5, v/v) and on Silica Gel G thin-layer plates in chloroform-methanol-water (65:25:4, v/v) and in chloroform-methanol-acetic acid-water (250:74:19:3, v/v). The synthetic reference phospholipids, cardiolipin, phosphatidylglycerol, phosphatidylethanolamine, and lysylphosphatidylglycerol, were kindly donated by G. H. de Haas and P. P. M. Bonsen of this laboratory. Furthermore, comparisons were made with the phospholipids of B. megaterium which had been identified previously (12, 13). Chromatograms were stained with: (i) the ninhydrin reagent for free amino groups, (ii) the periodate-schiff reagent for vicinal hydroxyl groups, (iii) the molybdate reagent for phosphate groups, (iv) the tricomplex staining method for lipids (6), (v) the alkaline silver nitrate reagent for sugars (15), and (vi) charring with 20% (v/v) H2SO4 on thin-layer plates. Hydrolysis of the phospholipids. Acid hydrolysis was cartied out with 4 N HCl at 100 C for 5 hr in a sealed tube. Amino acids were determined on a Beckman amino acid analyzer. Sugars were identified by paper chromatography with appropriate standards in n-butyl alcohol-acetic acid-water (6:1:2, v/v), n-propanol-concentrated ammonia-water (6: 3:1, v/v), and phenol-water (5:2, w/w). Strong alkaline hydrolysis was performed by the method of Benson and Maruo (1). The water-soluble products were investigated by a two-dimensional combination of high-voltage electrophoresis on paper (50 v/cm for 30 min in pyridineacetic acid-water (1:10:89, v/v, ph 3.6), followed by chromatography in propanol-concentrated ammoniawater (6:3:1, v/v). Mild alkaline hydrolysis of the aminoacyl phosphoglyceride was carried out according to Houtsmuller and van Deenen (9) in a buffered medium at ph 9.0. Hydrolysis of L-lysine was performed by means of L-lysine decarboxylase (EC ), which was purchased from Nutritional Biochemicals Corp. (Cleveland, Ohio). Preparation of protoplasts. Stationary-phase cells of B. subtilis were suspended in 0.06 M phosphate buffer (ph 6.2) containing 0.3 M sucrose. A 1-mg amount N-acetyl-muramide glucanohydrolase (Worthington Biochemical Corp.; EC ) per ml of suspension was added, and protoplast formation was examined by use of the phase-contrast microscope. Lysis of the protoplasts was obtained by suspending the protoplasts in distilled water and by ultrasonic treatment for 30 sec. The membrane fraction was isolated from this lysate by centrifugation at 26,000 X g for 30 min. The osmotic behavior of protoplasts was measured after diluting a concentrated protoplast suspension with a sucrose-containing phosphate buffer (0.06 M, ph 6.2), until an absorbance of at 550 nm was reached. The decrease in absorbance of protoplasts which were suspended in 0.15 M sucrose solutions and in distilled water was measured in a Unicam spectrophotometer at 550 nm. The amount of 260-nm absorbing material, released into the medium during lysis, was measured in the supernatant fraction after centrifugation at 30,000 X g for 15 min. RESULTS Identification of the phospholipids. B. subtilis cultivated in the two different media appeared to contain at least five different phospholipids (Fig. 1). To determine the identity of each compound, the total lipid extract was separated on a silica column. The neutral lipids were eluted from the column with chloroform but were not investigated in detail. The phospholipids were eluted from the column with increasing amounts of methanol-chloroform. The different fractions were investigated for their phospholipid content by thin-layer chromatography on microscope slides coated with silica, and similar fractions were pooled. By this procedure, three main fractions were obtained. They were eluted with 2%, 4 to 12%, and 20% methanol in chloroform and contained, respectively, compound 1, compounds 2 and 3, and compound 4. The phospholipid showing the lowest RF value on paper chromatograms appeared to be present in minor quantities and was not investigated further. Cardiolipin. Compound 1 was eluted with 2% methanol in chloroform and exhibited chromatographic properties identical to those of synthetic cardiolipin. Alkaline hydrolysis of this compound gave rise to the formation of bis-(glycerylphosphoryl) glycerol as the only water-soluble product (Fig. 2). Both observations confirm the identity of compound 1 with cardiolipin. Phosphatidylethanolamine and phosphatidylglycerol. Elution of the column with 4 to 12% methanol in chloroform gave mixtures of compounds 2 and 3. Separation of both phospholipids was obtained by preparative thin-layer chromatography on Silica Gel G plates in chloroform-methanol-water (65:25:4, v/v). Compound 2 exhibited chromatographic properties identical to the reference phosphatidylethanolamine, and alkaline hydrolysis of this compound yielded glycerylphosphorylethanolamine (Fig. 2). The staining properties of compound 3 (ninhydrin-

3 300 OP DEN KAMP, REDAI, AND VAN DEENEN J. BACTERIOL FiG. 1. Autoradiogram of32p-labeled phospholipids of B. subtilis grown in alkaline broth (a) and alkaline broth plus 2% glucose and 0.2% ammonium sulfate (b). The lipid extracts were separated on silica gel-impregnated paper with the solvent system of Marinetti et al. [(11); diisobutyl ketone-acetic acid-water (40:25:5, v/v)]. The phospholipids were identified as: (1) cardiolipin; (2) phosphatidylethanolamine; (3) phosphatidylglycerol; (4) lysylphosphatidylglycerol; (5) unknown. + - Electrophoresis - GPGPG 0 0Origin LysO oegpl GPG 0 GPE FIG. 2. Separation of alkaline hydrolysis products of phosphoglycerides. Electrophoresis was carried out in the horizontal direction in pyridine-acetic acid-water (1:10:89, v/v; ph 3.6) for 30 min at 50 v/cm. Further separation was obtained by chromatography in the second direction in propanol-concentrated ammoniawater (6:3:1, v/v). Abbreviations: GPGPG, bis- (glycerylphosphoryl)glycerol; agp, a-glycerophosphate; GPG, glycerylphosphorylglycerol; GPE, glycerylphosphorylethanolamine; and Lys, lysine. negative, periodate-schiff-positive) had already indicated the identity of this phosphoglyceride with phosphatidylglycerol. This observation was confirmed by alkaline hydrolysis, which resulted in the formation of glycerylphosphorylglycerol. Glucolipid. Chromatographic investigations of the column fractions, discussed above, revealed the presence of a nonphosphorus-containing lipid. The compound could be isolated by preparative thin-layer chromatography in chloroform-methanol-water (65:25:4, v/v) and was hydrolyzed in 4 N HCI at 100 C for 5 hr. The water-soluble products were investigated with paper chromatography by use of the solvent systems described above. When stained with the alkaline silver nitrate reagent and the periodate-schiff reagent, the chromatograms exhibited two spots having RF values identical to those of glycerol and glucose. These observations are in agreement with the findings of Brundish et al. (5) and Bishop et al. (2), who demonstrated the presence of a diglucosyldiglyceride in B. subtilis, and no attempt was made to study the structure of the compound in more detail. Lysylphosphatidylglycerol. Compound 4 was eluted from the column with 20% methanol in chloroform, and the chromatographic and staining properties of this compound were found to be identical to those of lysylphosphatidylglycerol isolated from B. megaterium (12) and lysylphosphatidylglycerol obtained by chemical synthesis (4). On alkaline hydrolysis according to Benson and Maruo (1), the purified compound yielded glycerylphosphorylglycerol (Fig. 2), whereas treatment of the compound with a borate buffer (ph 9.0) resulted in the formation of phosphatidylglycerol and free lysine. After acid hydrolysis of thle column fraction, which contained this phospholipid, 80% of lysine and small amounts of several other amino acids were found. To investigate the possibility that other aminoacylphosphoglycerides were present in addition to the lysylphosphatidylglycerol, the fraction was treated as follows. Part of the lysylphosphatidyl-

4 VOL. 99, 1969 PHOSPHOLIPID COMPOSITION OF B. SUBTILIS glycerol-containing fraction was taken up in a small amount of chloroform, applied on a paper, and subjected to high-voltage electrophoresis at 50 v/cm for 30 min in pyridine-acetic acid-water (1:10:89, v/v). The edges of the paper were sprayed with ninhydrin to reveal the lipid and other amino nitrogen-containing material. Apart from the lysylphosphatidylglycerol which remained at the origin, two compounds could be detected; these compounds migrated towards the negative pole, reacted strongly with ninhydrin, but did not contain phosphorus (Fig. 3). All of the compounds were eluted from the paper with the appropriate solvents, hydrolyzed in 4 N HCI at 100 C for 5 hr, and investigated for their amino acid composition. Lysine constituted 95% of the amino acids recovered from the spot of lysylphosphatidylglycerol, whereas the other nonlipid compounds contained all of the amino acids normally found in proteins. (The mobility of both compounds in the electrophoretic system and their solubility in water make it unlikely that these compounds are lipid in nature. The presence of very small amounts of other amino acid-containing lipids in the lysylphosphatidylglycerol fraction cannot be excluded, and more experiments have to be carried out to solve this problem.) Furthermore, the lysylphosphatidylglycerol which was purified by high-voltage electrophoresis appeared to have a phosphorus to lysine ratio of 1.1. These results indicated that the amino acids which are found after hydrolysis of chromatographically isolated lysylphosphatidylglycerol are, apart from lysine, not covalently linked to a phosphoglyceride but originate from contaminating material. Finally, the complete conversion of lysine into cadaverine by means of a specific L-lysine decarboxylase demonstrated that only the L-isomer of lysine was present. Influence of the growth medium on the phospholipid composition. The phospholipid composition of B. subtilis was shown to depend on the PG +I I 2 3 Origin Lysine FIG. 3. Purification of lysylphosphatidylglycerol. The phospholipid was solubilized in a small volume ofchloroform, applied on the paper and subjected to highvoltage electrophoresis in pyridine-acetic acid-water (1:10:89, v/v; ph 3.6), for 30 min at 50 v/cm. In a control experiment, phosphatidylglycerol and lysine were mixed in the same buffer and applied on the paper. Compounds are: (1) lysylphosphatidylglycerol; (2) and (3) nonlipid, amino nitrogen-containing material. PG, phosphatidylglycerol. culture conditions in a similar way as has been shown to occur in S. aureus (8) and B. megaterium (13). Cells of B. subtilis which were cultivated in medium A contained 3.7% of lipid, 38% of which was phospholipid, the rest being neutral lipid and glycolipid. Addition of glucose and (NH4)2SO4 to the culture medium (medium B) did not influence the lipid content (3.6%) to a great extent, and the amount of phospholipid (30% of the total lipid) remained fairly constant (Table 1). The phospholipid pattern, however, appeared to be changed considerably after growth in medium B. The amount of phosphatidylglycerol, which was high (36% of the total phospholipids) in cells which were harvested from medium A, was found to be diminished markedly in cells grown in medium B. This alteration in the phosphatidylglycerol content appeared to be counterbalanced for the greater part by a shift in the amount of lysylphosphatidylglycerol. Cultivation of the bacteria in medium B nearly doubled the absolute amount of lysylphosphatidylglycerol per gram (dry weight) compared with cultivation in medium A. The amounts of the other phospholipids, cardiolipin and phosphatidylethanolamine, remained fairly constant under the different conditions. It has been demonstrated (9, 13) that the shift in phospholipid composition in S. aureus and B. megaterium is due, at least partly, to a difference in the acidity of the culture medium during growth of the bacteria. A similar effect of the ph on the phospholipid composition is proposed for B. subtilis. Growth of the bacteria at neutral ph resulted in a high ratio of phosphatidylglycerol to lysylphosphatidylglycerol, whereas a greater acidity of the medium, induced TABLE 1. Lipid Lipid content of B. subtilis' Per cent of dry weight Per cent of total phospholipid Medium Medium Medium Medium A B A B Total lipid Total phospholipid Cardiolipin Phosphatidylethanolamine Phosphatidylglycerol Lysylphosphatidylglycerol a The ph at harvesting was 7.0 for medium A and 5.5 for mediun} B.

5 302 OP DEN KAMP, REDAI, AND VAN DEENEN J. BACTERIOL. *100 c u to D0 m a.0 _ 40 0,X 60 CL by the fermentation of glucose, increased the amount of the Zwitter-ionic phosphoglyceride at the cost of the phosphatidylglycerol content. Prostoplasts of B. subtilis. Cells of B. subtilis harvested from the two different media were converted into protoplasts by enzymatic removal of the cell wall. During examination of the conversion of intact cells into protoplasts by use of phase-contrast microscopy, similar observations were made as have been described previously for B. megaterium (13). Rod-shaped cells of B. subtilis which were grown in medium A were rapidly converted into spherical protoplasts, whereas cells grown in an acidic environment tended to maintain their original cell shape to a great extent during the treatment with lysozyme. The latter protoplasts appeared to have lost the whole cell wall despite their rod-shaped form (10). The protoplasts which were obtained from cells grown at low ph displayed a deviant behavior in hypotonic media, as compared with the spherical protoplasts derived from cells which were harvested at neutral ph (Fig. 4). Lowering the tonicity of the medium in which the protoplasts were prepared resulted in lysis of the protoplasts, as could be shown by a decrease in the optical density. Whereas the protoplasts made from cells grown in medium A (ph 7.2) showed a rapid lysis, the rod-shaped protoplasts seemed to be resistant to a decrease in the sucrose concentration (Fig. 4). Measurements of the optical density at 260 nm, however, demonstrated that both spherical and rod-shaped protoplasts released their cellular content in the medium 20'- 01 ~ ~ ~ ~ Time (min.) FIG. 4. Behavior of B. subtilis protoplasts in a hypotonic medium. Protoplasts, preparedfrom cells grown at neutral ph (A) and in an acidic environment (0), were suspended in a hypotonic solution of0.15 M sucrose in 0.06 m phosphate buffer, ph 6.2. The decrease of the absorbance was measured at 550 inm. For comparison, the behavior of B. megaterium protoplasts, grown at low ph under similar conditions, has been shown (dotted line; see reference 13). under hypotonic conditions (Fig. 5). Therefore, the experiments indicate that in a hypotonic medium lysis of rod-shaped protoplasts occurs but that the remaining structures retain the original cell form to a great extent. DISCUSSION The phospholipids which have been identified in B. subtilis appear typical of those found to occur in other bacilli investigated thus far. B. megaterium (12), B. licheniformis (14), and B. cereus (9) have been shown to contain a mixture of cardiolipin, phosphatidylglycerol, phosphatidylethanolamine, and aminoacyl phosphoglycerides. These phospholipids can also be detected in B. subtilis, as has been described above and in a recent publication of Bishop et al. (2). The nature of the lipoamino acid was investigated by these authors, who showed that acid hydrolysis of the lipoamino acid fraction yields mainly lysine (80%) but that smaller amounts of alanine (7%), glycine (5%), and leucine (3%) can also be detected. The aminoacyl phosphatidylglycerol that we isolated by chromatography gave, after acid hydrolysis, about 80% of lysine and a whole variety of other amino acids. Further purification by means of high-voltage electrophoresis, however, indicates that the other amino acids probably originate from associated material which is not covalently bound to the phosphatidylglycerol. Certain differences can be noted between the quantitative data on the lipid content of B. subtilis obtained by Bishop et al. (2) and the results presented here. The ratio of phospholipid to total lipid in the lipid extracts from B. subtilis, which was nearly 0.4 in our experiments, was found to,wuu. E coo 0 " '0 c lhoo.0 6n fi. -i Time (min) FiG. 5. Release of intracellular material from B. subtilis. Protoplasts which were prepared from cells grown at neutral ph (0) and at low ph (A) were suspended in 0.15 M sucrose-containing phosphate buffer (0.06 M, ph 6.2). After incubation, the residual protoplasts and membranes were centrifuged at 26,000 X g for IS min, and the absorbance of the supernatant fluid was measured at 260 nm. A control experiment was carried out in an isotonic sucrose solution (0.3 i; dashed line).

6 VOL. 99, 1969 PHOSPHOLIPID COMPOSITION OF B. SUBTILIS 303 be 0.75 by Bishop et al. (2). It should be mentioned, however, that in the latter experiments polyhydroxybutyric acid was removed before the total amount of lipid was estimated. As mentioned by Bishop et al., up to 16% of the whole cell lipid can consist of polyhydroxybutyric acid, which accounts for the observed differences in the phospholipid to total lipid ratio. The phospholipid composition of several grampositive organisms was found to be influenced by the culture conditions, especially by the acidity of the medium during growth (9). The results obtained with B. subtilis are consistent with this observation (Table 1). In this bacterium, however, the ph-dependent decrease in the phosphatidylglycerol content and the increase in the amount of its lysyl derivative were less pronounced, as was observed in S. aureus (9). A possible explanation can be offered by the observation that growth of B. subtilis in a glucose-containing medium induced a decrease in the ph from 7.0 to 5.5, whereas S. aureus, cultivated under the same conditions, lowered the ph to 4.8. Experiments with S. aureus and with B. megaterium have shown that a small decrease in ph is accompanied by a minor shift in phospholipid composition only. The small amount of lipoamino acids found by Bishop et al. (2) may have resulted from the use of a buffered growth medium (ph 7.6). Growth of B. subtilis in an acidified medium influenced the shape and the osmotic behavior of the protoplasts of these organisms in a similar way as has been described for B. megaterium (13). Lysozyme treatment of cells which had been exposed to a low ph resulted in the formation of protoplasts which appeared to have maintained the original cell shape to a great extent. The lysis of such protoplasts, which was achieved by lowering the tonicity of the medium, resulted in a reduction in the decrease in the optical density at 550 nm, as compared with protoplasts made from cells which were harvested from a neutral medium. Measurements of the release of intracellular material when both kinds of protoplasts were suspended in a hypotonic medium indicated, however, that rod-shaped protoplasts are also unstable under hypotonic conditions. These observations demonstrate that, under hypotonic conditions, the protoplasts which have been derived from cells grown at low ph maintain, despite their loss of intracellular material, a structural integrity which is responsible for the absorbance at 550 nm and which may result from a more rigid membranous structure, as compared with protoplasts made from cells which were cultivated at neutral ph. The latter phenomenon, however, appeared to be less pronounced, as was observed with protoplasts from B. megaterium. Growth of B. megaterium in the glucose-containing medium resulted in a ph at harvesting of 4.8 to 5.0, whereas under similar conditions B. subtilis was harvested from a medium of ph 5.5 Since it was shown that the ph of the medium is closely related to the observed changes in membrane composition and properties of the protoplasts, it can be argued that a less pronounced decrease in ph during growth will be accompanied by less pronounced alterations in both the phospholipid composition and the behavior of the protoplasts (without suggesting a direct relationship between both phenomena). ACKNOWLEDGMENTS The present investigation was carried out under the auspices of The Netherlands Foundation for Chemical Research (S.O.N.) and with financial aid from The Netherlands Organization for the Advancement of Pure Research (Z.W.O.). The assistance of M. T. Kauerz is gratefully acknowledged. LITERATURE CITED 1. Benson, A. A., and B. Maruo Plant phospholipids. I. Identification of the phosphatidyl glycerols. Biochim. Biophys. Acta 27: Bishop, D. G., L. Rutberg, and B. Samuelsson The chemical composition of the cytoplasmic membrane of Bacillus subtilis. Eur. J. Biochem. 2: Bligh, E. G., and W.-J. Dyer A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37: Bonsen, P. P. M., G. H. de Haas, and L. L. M. van Deenen Synthetic and structural investigations on 3-phosphatidyl-1'-(3'-O-L-lysyl)glycerol. Biochemistry 6: Brundish, D. E., N. Shaw, and J. Baddiley The occurrence of glycolipids in gram-positive bacteria. Biochem. J. 95:21c-22c. 6. Bungenberg de Jong, H. G Improved detection of phosphatides on paperchromatograms with a mixed staining solution. Proc. Koninkl. Ned. Akad. Wetensch. Ser. B Phys. Sci. 64: Fiske, C. H., and Y. SubbaRow The colorimetric determination of phosphorus. J. Biol. Chem. 66: Houtsmuller, U. M. T., and L. L. M. van Deenen On the accumulation of amino acid derivatives of phosphatidylglycerol in bacteria. Biochim. Biophys. Acta 84: Houtsmuller, U. M. T., and L. L. M. van Deenen On the amino acid esters of phosphatidylglycerol from bacteria. Biochim. Biophys. Acta 106: Iterson, W. van, and J. A. F. Op den Kamp Bacteriashaped gymnoplasts (protoplasts) of Bacillus subtilis. J. Bacteriol. 99: Marinetti, G. V., J. Erbland, and J. Kochen Quantitative chromatography of phosphatides. Fed. Proc. 16: Op den Kamp, J. A. F., U. M. T. Houtsmuller, and L. L. M. van Deenen On the phospholipids of Bacillus megaterium. Biochim. Biophys. Acta 106: Op den Kamp, J. A. F., W. van Iterson, and L. L. M. van Deenen Studies on the phospholipids and morphology of protoplasts of Bacillus megaterium. Biochim. Biophys. Acta 135: Rogers, H. J., D. A. Reaveley, and J. D. J. Burdett The membrane systems of Bacillus licheniformis, p In H. Peeters (ed.), Protides of the biological fluids, vol. 15. Elsevier Publishing Co., Amsterdam. 15. Trevelyan, W. E., D. P. Proctor, and J. S. Harrison Detection of sugars on paper chromatograms. Nature 166: