JOURNAL OF BACTERIOLOGY, Jan. 1976, p. 300-307 Copyright i 1976 American Society for Microbiology

Vol. 125, No. 1 Printed in U.S.A.

Effect of Alkali on the Structure of Cell Envelopes of Chlamydia psittaci Elementary Bodies TOSHIHIKO NARITA, PRISCILLA B. WYRICK,

AND

G. P. MANIRE*

Department of Bacteriology and Immunology, School of Medicine, University of North Carolinia, Chapel Hill, North Carolina 27514 Received for publication 29 September 197.5

Suspensions of isolated cell envelopes of infectious elementary bodies (EB) of Chlamydia psittaci at alkaline pH showed a rapid, extensive decrease in absorbance, accompanied by the release of a cell envelope component in a sedimentable form. This phenomenon was observed both at 0 C and with envelopes which had been previously heated to 100 C. Monovalent and divalent cations effectively inhibited the turbidity loss, whereas ethylenediaminetetraacetate (EDTA) caused an accelerated decrease in turbidity. The turbidity loss observed after incubation of the envelopes at alkaline pH could be reversed to the level of the initial value by dialysis against distilled water containing Mg2+. Thin-section electron photomicrographs of purified EB exposed to alkaline buffer with EDTA revealed the loss of the internal contents of cells, but these cells still maintained their round shapes. The cell surface of treated EB appeared pitted in negatively stained preparations, whereas intact EB had a smooth surface. Electron microscopic studies on negatively stained preparations of the clear supernatant obtained after the treatment of the envelope with alkaline buffer containing EDTA demonstrated the presence of spherical particles, approximately 6 to 7 nm in diameter, and rodlike particles, which appeared to be made up of two or more spherical particles.

Chlamydiae vary in morphology during their developmental cycle, with alternation between the infectious, rigid elementary body (EB) and the noninfectious, fragile reticulate body (RB). The cell surface structures, or envelopes, of these two forms have been reported to be similar, both morphologically and in cell wall amino acid analysis, to those of gram-negative bacteria such as Escherichia coli (15, 24, 27). However, the inability to detect muramic acid in purified EB or their cell walls (9, 15) and the failure of lysozyme-ethylenediaminetetraacetate (EDTA) to effect the integrity of the envelopes of Chlamydia (25) indicate that the cell envelopes of Chlamydia differ fundamentally from those of most bacteria. Thus, the question arose as to whether the chlamydial cell surface structures maintain their organization in the same way as walls of gram-negative or gram-positive bacteria. Whereas lability of cell envelopes, especially external envelope components, of gram-negative bacteria is well known (5, 8), there are no reports on such properties of chlamydial walls, namely, the events leading to dissolution or disintegration of the cell surface constituents. We have found that exposure of a suspension of

isolated cell envelopes of EB to alkaline buffer results in an extensive decrease in absorbance, analogous to behavior of gram-negative bacterial envelopes under similar conditions. The present paper describes the effect of alkaline buffer on cell envelopes of EB. MATERIALS AND METHODS Organisms and cultivation. The Cal 10 meningopneumonitis strain of Chlamydia psittaci was used throughout these experiments. The organisms were grown in L-cell suspension cultures according to the method of Tamura and Higashi (23). L-cells in suspension culture were routinely propagated in 0.01% yeast extract-0.5% lactoalbumin hydrolysateEarle balanced salt solution (YLE; GIBCO, New York, N.Y.) on rotory shakers or in spinner flasks. For infection, the cells were then transferred to a 14-liter fermenter and infected with meningopneunonitis organisms. Equal volumes of the culture and fresh medium were incubated at 37 C for 40 to 44 h. Growth of meningopneumonitis organisms in cells was monitored by Giemsa- or Macchiavello-stained smears. Isolation of purified EB and RB. EB were prepared and purified as previously reported (23), except trypsin treatment was not used in the purification of EB for our studies. Preparation of purified RB followed the methods described previously (26). Preparation of cell envelope of EB. Cell envelopes 300

EFFECT OF ALKALI ON C. PSITTACI

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301

of EB were prepared from purified suspensions of EB according to the method of Manire and Tamura (15). Measurement of absorbance change. EB or RB suspended in distilled water were adjusted to an absorbance of approximately 4.0 at 550 nm, and the suspension was divided into 1.0-ml portions and stored at -70 C. Prior to use, this stock suspension was thawed and diluted with an appropriate amount of chilled distilled water so that, when added to the incubation tube containing buffer, there was a starting adjusted absorbance of about 0.5 at 550 nm. The change in turbidity was routinely followed by measuring the decrease of the absorbance at 550 nm with a Gilford model 240 spectrophotometer. Measurement of the turbidity change of suspensions of isolated cell envelopes was performed essentially the same as that for the whole cells. To measure the release of ultraviolet (UV)-absorbing substances from cells, the cell suspension was centrifuged at 30,000 x g for 10 min at 4 C, and the clear supernatant fluid was read at 260 nm. Analysis of protein content. The protein was determined by the Folin phenol method of Lowry et al. (14), with bovine serum albumin (Sigma Chemical Co., St. Louis, Mo.) as a reference. Electron microscopy. Sample pellets for thin-section electron microscopy were prefixed in 2.5% glutaraldehyde prepared in 0.01 M sodium cacodylate buffer for 1 h at 4 C, enrobed in agar blocks, and washed thoroughly in 0.135 M sodium cacodylate. Primary fixation was accomplished by 1% OSO4 prepared in veronal-acetate buffer, pH 7.3, for 90 min at 25 C, followed by postfixation with uranyl acetate for 60 min in the dark. The samples were dehydrated in acetone and embedded in Vestopal W. Thin sections were cut on a Reichert ultramicrotome and poststained with uranyl acetate (28) for 15 min and lead citrate (20) for 4 min. Identical samples were stained negatively with 2% phosphotungstic acid (pH 7.2) solution (16). All specimens were examined with an AEI electron microscope, accelerated at 60 kV, with a 30-gm objective aperture.

pH 8.0, employed. Varying the concentration of carbonate buffer has no noticeable effect on the results. EB suspended in distilled water or phosphate-buffered saline (pH 7.41 ex¶hibited a 15% turbidity loss in 24 h of incubation. The effect of the temperature of incubation at alkaline pH was studied. Incubation of EB in

0

801F

RESULTS The effect of incubating EB for 2 h in 0.05 M buffers of varying pH values is illustrated in Fig. 1. Five buffers with overlapping pH ranges were employed. Little turbidity loss was apparent below pH 8.0, and clumping of cells was observed below pH 6.0. Maximum turbidity loss occurred between pH 10 and 12. Leakage of UV-absorbing substances into the cell supernatant coincided with the decrease in turbidity of the suspension. Incubation for 72 h in buffers above pH 9.0 resulted in a maximum turbidity loss of 70%. Figure 2 represents the effect of buffer molarity on the rate of decrease in absorbance of the EB suspension. Sodium carbonate-bicarbonate (carbonate) buffer, pH 10, sodium phosphate (phosphate) buffer, pH 8.0, or tris(hydroxymethyl)aminomethane (Tris)-hydrochloride buffer,

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pH FIG. 1. Effect of pH on stability of whole EB. Turbidity loss and the leakage of UV-absorbing substances of suspension of EB after 12 h of incubation at 38 C in 0.05 M buffers were examined at different pH values. Symbols: *, citrate buffers (pH 4 to 6); 0, phosphate buffers (pH 6 to 8.5); 0, Tris buffers (pH 8 to 9); 0, carbonate buffers (pH 9 to 10.5); A, borate buffers (pH 10.5 to 12); 0-- -0, absorption of cellfree supernatant at 260 nm.

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BUFFER CONCENTRATION (m M) FIG. 2. Effect of buffer concentration on the stability of whole EB. Turbidity loss of suspension of EB after 12 h of incubation at 38 C was examined in buffers of different molarities. Symbols: 0, carbonate buffers (pH 10); 0, phosphate buffers (pH 8); U, Tris buffers (pH 8).

302

NARITA, WYRICK, AND MANIRE

J. BACTERIOL.

0.01 M carbonate buffer, pH 10, at 37 C showed the same extent of decrease in turbidity observed at 20 or 0 C (Fig. 3). The higher the incubation temperature (0 to 37 C) with pH 8.0, 0.01 M phosphate buffer, the more rapid and extensive was the turbidity loss. However, no remarkable increase in turbidity loss was observed above 37 C. The suspension of EB was essentially stable at 0 C, and no leakage of UV-absorbing substances was found. Figure 4 indicates the effect of various compounds on the leakage of UV-absorbing substances, used to monitor the instability of EB. Divalent cations were more effective than monovalent cations in retarding the leakage of UV-absorbing substances. K+ plus Li+ and Mg2+ could be substituted for Na+ and Ca2+, respectively. The rate of release of UV-absorbing substances from cells was accelerated by the presence of EDTA. A hypertonic solution of sucrose (0.5 M) was not effective in preventing turbidity loss or the release of UV-absorbing substances from EB suspensions, suggesting that the osmotic pressure plays only a small role in cellular stability under these conditions. Instead, the data indicate that the instability of EB of C. psittaci in alkaline buffer may be related to the behavior of ionizable groups in the EB envelopes. Preliminary experiments demonstrated that suspensions of whole RB also showed the same turbidity changes at alkaline pH. For all subsequent experiments, the standard assay consisted of suspending isolated EB cell envelopes in pH 10 carbonate buffer (0.01 M). A

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FIG. 4. Factors affecting the leakage of UV-absorbing substances from whole EB. EB were incubated at 38 C in pH 10, 0.01 M carbonate buffer alone or in buffer plus indicated supplement. Symbols: 0, buffer alone; 0, 0.001 M EDTA; 0, 0.5 M sucrose; *, 0.5 M Na+; A, 0.01 M Ca2+. Cell-free supernatant was assayed at 260 nm for the leakage of UV-absorbing substances.

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rapid turbidity loss, at incubation temperature of 38 or 0 C, was observed to the same extent as that in whole cells. (Fig. 5). Preheating the envelopes at 100 C for 10 to 30 min partially inhibited turbidity loss, perhaps due to nonspecific coagulation of envelope proteins. The effect of EDTA, Na+, and Ca+ on turbidity loss of suspensions of EB cell envelopes at pH 10 was examined, and the results are shown in Fig. 6. When the envelopes were incubated at

1

2

3

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TIME (HOURS) FIG. 3. Effect of temperature of incubation on the stability of whole EB. Turbidity loss of suspensions of whole EB at different temperatures of incubation. Symbols: (0) 0, 20, or 38 C in pH 10 carbonate buffer; (0) 38 C in pH 8 phosphate buffer; (U), 0 C in pH 8 phosphate buffer.

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TIME (HOURS) FIG. 5. Turbidity loss of suspensions of isolated EB cell envelopes. Symbols: 0, intact envelopes incubated at 38 C in pH 10 carbonate buffer; intact envelopes incubated at 0 C in pH 10 carbonate buffer; 0, cell envelopes preheated for 10 min at 100 C and incubated at 38 C in pH 10 carbonate buffer. U,

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TIME (HOURS) FIG. 6. Factors affecting turbidity loss of suspensions of isolated EB envelopes. Isolated cell envelopes were exposed to pH 10, 0.01 M carbonate buffer alone or the buffer plus indicated supplement. Symbols: 0, buffer alone; 0, 0.01 M EDTA; *, 0.5 M Na+; A, 0.01 M Ca2

303

EFFECT OF ALKALI ON C. PSITTACI

VOL. 125, 1976

sedimented by centrifugation at 110,000 x g for a Spinco SW39L rotor. These results indicated that exposure of isolated EB envelopes to alkaline pH resulted in the liberation of envelope constituents, which contain protein in particulate form. The various effects of pH and cations and the susceptibility to EDTA of EB cell envelopes all suggest that ionic interactions are involved in the maintenance of intact cell envelopes of EB. It seems likely that the envelopes contain ionic linkages which are weakened or disrupted by alkali, with a subsequent loss of some components. To determine if the turbidity loss was a reflection of the dissociation of envelope components by electrostatic repulsion between negatively charged groups, as observed in the enve3 h in

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38 C in pH 10 carbonate buffer containing 1 mM EDTA, the decrease in absorbancy was immediate and the rate of decrease was accelerated, whereas the turbidity loss was decreased in the presence of 0.5 M Na+ and almost completely inhibited by 0.01 M Ca+. After incubation of the envelopes in pH 10 carbonate buffer with 1 mM EDTA for 6 h at 38 C, the reaction mixture was dialyzed at 4 C for 18 h against distilled water with and without 10 mM Mg2+. Turbidity readings returned nearly to the original level on dialysis, and the presence of Mg2+ appeared to increase the speed and completeness of the reversal (Fig. 7). Dialysis against buffers at a pH of less than 10 was effective to various degrees in the reversal of the turbidity loss. If the reaction mixture after dialysis against distilled water alone was again exposed to pH 10 buffer, extensive turbidity loss was observed again. The fate of some of the envelope components released during alkali treatment was investigated. Intact cell envelopes could be sedimented by centrifugation at 12,000 x g for 1 h, but approximately 15% of the total envelope protein was no longer sedimentable by centrifugation at 12,000 x g for 1 h after exposure for 1 h to pH 10 carbonate buffer (Table 1). Release of protein components from the envelopes was almost complete within the first hour. Under higher gravitational forces, however, most of the protein liberated into the suspending buffer was recovered in the sediment. About 98% of the total amount of released envelope protein was

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TIME (HOURS) FIG. 7. Reversal of the turbidity loss of suspensions of EB cell envelopes. Isolated cell envelopes were exposed to 0.01 M carbonate buffer (0). After 6 h of incubation at 38 C, the suspension was dialyzed against distilled water (O- -U), or distilled water containing 0.01 M Mg2+ (O--- -0). After dialysis against distilled water alone, the suspension was then incubated as such (0) or again exposed to pH 10 carbonate buffer (U). -

TABLE 1. Release of EB cell envelope protein during alkali exposurea Incubation time (h)

Turbidity

0 1 12

0 57 66

loss

Released protein (%)b SupIc Sup-IId

0.1 14.6 15.4

0.1 2.0 2.1

aThe envelopes were exposed to pH 10, 0.01 M carbonate buffer at 38 C. b Expressed as percentage of released protein/total envelope protein. Value given represents the mean of values obtained for two different experiments. c Supernatant fluid after centrifugation twice for 1 h at 12,000 x g at 4 C. d Supernatant fluid after centrifugation for 3 h at 110,000 x g at 4 C.

304

NARITA, WYRICK, AND MANIRE

lope dissolution of some of gram-negative bacteria (2), experiments were designed to examine the effect of alteration of a number of free carboxyl groups in the envelope (3, 11, 22). Removal of potential negative changes by addition of glycine methyl ester, removal of one negative charge and the addition of another positive charge by addition of an ethylenediamine radical, and an increase in the number of free carboxyl groups by succinylation all failed to show any significant effects on the rate and extent of absorbance decrease. Attempts to implicate lytic enzymes in the instability of cell envelopes were also made. Suspensions of intact EB or isolated envelopes were incubated at 38 C for 12 h in alkaline buffer. The cell-free supernatant fluid, the cell debris, and the supernatant fluid from an EB suspension treated with high concentrations of salt at 0 C, which reportedly removes autolytic enzymes effectively (7), were added to intact or heated envelopes. None of these preparations caused any significant changes in turbidity loss. Thus, it appeared unlikely that the decrease in absorbance was attributed to the formation or release of lytic enzymes. The effects on structure of EB by treatment with alkaline buffer were examined by electron microscopy. Whole cells or cell envelopes of EB were incubated for 12 h at 38 C in 0.01 M carbonate buffer (pH 10) with 1 mM EDTA. After incubation, the suspensions were centrifuged at 12,000 x g for 1 h, and the precipitates were processed for transmission electron microscopy. Figures 9 and 10 represent thin-section electron photomicrographs of the treated whole cells and cell envelopes. When compared to control cells (Fig. 8), the intact whole EB incubated in alkaline buffer at pH 10 appear to have lost their internal contents (Fig. 9). It should be noted that treated cells and cell envelopes appear to have retained their rigid, round shapes. Negatively stained preparations of control and treated whole cells are shown in Fig. 11 and 12. The cell surface of the treated EB appeared granular and occasionally "pitted" (Fig. 12), whereas the cell surface of the untreated EB (Fig. 11) appeared smooth. These results suggested the liberation of cell surface components by alkali treatment. The cell-free supernatant fluid obtained after centrifugation at 12,000 x g for 1 h was again centrifuged under the same conditions, and the resulting supernatant fluid was in turn centrifuged at 30,000 x g for 1 h at 4 C.The clear 30,000 x g supernatant fluid was dialyzed for 72 h at 4 C against several changes of distilled water and lyophilized. The lyophilized preparation was

J. BACTERIOL.

reconstituted in pH 7 phosphate buffer and examined in the electron microscope after negative staining (Fig. 13). The supernatant preparation contained spherical particles, approximately 6 to 7 nm in diameter, and rodlike particles, which appear to be made up of two or more spherical particles. The particles may have been released orginally as a spherical unit, but on lyophilization and reconstitution in buffer of lower pH aggregation may have resulted. Thus, it seems that exposure of isolated cell envelopes to alkaline buffer causes the release of spherical particles, and that these particles form a significant part of the cell envelope. DISCUSSION Incubation of EB of C. psittaci in pH 10 carbonate buffer results in the release of UVabsorbing substances and the subsequent appearance of empty envelopes without complete disruption of cell surface structures. The data indicate that the alkali treatment causes an alteration in the permeability barrier, which is not prevented by suspending the cells in an osmotically protective environment. It is well known that the outer membrane of the gram-negative cell envelopes constitutes a barrier which is breached by the Tris-EDTA system (5). Such treatment of gram-negative bacteria partially liberates cell wall lipopolysaccharide and causes a transient, nonspecific increase in permeability (13). The effect of alkali on EB appeared to be similar in some respects to the action of polymixin B on gram-negative bacteria (18). Polymixin B causes changes in the permeability barrier of cell surface structures of gram-negative bacteria. The antibiotic acts on the lipopolysaccharide portion in the envelope to form numerous projections on cell surfaces through which leakage of the cellular contents occurs (12). Matsumoto et al. (16) demonstrated that purified cells or cell walls on EB or RB treated with polymixin B were covered with a number of projections, and that the intracellular components were greatly disturbed. Their results and those presented in this paper suggest that the external layer of the cell envelopes of EB does bear similarity to those of typical gram-negative bacteria, and substances resembling the lipopolysaccharide of gram-negative species may exist in the envelope of EB. Electron microscopic studies on the supernatant fluid after alkali treatment revealed the presence of spherical and rodlike particles. Release of these particles from the cell envelope resulted in a pitted appearance of the envelope as viewed in the electron microscope. The exact location

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FIG. 8-12. Transmission electron photomicrographs of intact EB or isolated EB envelopes before and after treatment in pH 10 carbonate buffer. Bars, 0.1 tim. FIG. 8. A thin section of intact EB (control). Each cell is surrounded by an envelope consisting of two

membrane layers. FIG. 9. A thin section of alkali-treated EB. Despite the loss of intracellular contents, the cells maintain their rigid, round shapes. FIG. 10. A thin section of alkali-treated, isolated cell envelopes of EB. The envelopes retain their rigid shapes. FIG. 11. A negatively stained preparation of intact EB (control). The cell surface appears uniform and smooth. FIG. 12. A negatively stained preparation of alkali-treated EB. The cell surface appears granular and pitted. 305

306

NARITA, WYRICK, AND MANIRE

13 FIG. 13. Electron photomicrograph of a negativestained preparation of the lyophilized supernatant from an alkali-treated suspension of EB envelopes. Numerous spherical and rodlike particles are present (arrows). Bar, 0.1 um.

J. BACTERIOL.

of Mg2+, these particles were reconstituted in their original location in the wall. It was not determined in our investigation if the reversal of the turbidity loss, promoted by dialysis against Mg2+, was attributed to reassembly of the "released" materials or to their nonspecific aggregation. Electron photomicrographs of whole EB and isolated cell envelopes after treatment with alkali and EDTA revealed that these structures still retained rigidity. The mucopeptide, identified as the thin, electron-dense layer located between the outer membrane and cytoplasmic membrane in gram-negative bacterial envelopes (8), is believed to be responsible for the rigidity of these bacterial cell walls. This thin layer was not apparent in thin sections of EB or the isolated envelopes. Studies on C. psittaci, using methods that detect less than 1 ,ug of muramic acid, have failed to show its presence in cell walls (9). It may be concluded, therefore, that, even if the murein component exists in the wall, it is present in minute amounts and its occurrence in the envelope is atypical, and perhaps even its location is different. Buckmire and MacLeod (4) suggested that the murein layer in the envelope of a marine pseudomonad might be held intact by ionic bonds rather than by covalent linkages. If such were the case regarding the murein of EB, one would expect the cells to lose their rigid, round shapes during the treatment with alkali and EDTA, but they do not. The structures involved in the rigidity of EB envelopes may be elucidated by studying cell envelope residues obtained after treatment of the envelopes with alkali and EDTA, since the walls of gram-negative bacteria have been reported to be rendered sensitive to lysozyme after lipopolysaccharide removal from the envelopes by EDTA (19). Whereas chlamydial EB and RB differ in a number of respects, these studies have shown that they behave similarly in their response to alkali treatment, although there are differences in the composition of the cell envelopes of these two forms (15, 24).

of these particles in the envelope of EB is yet to be determined. The results of the present study indicate that ionic linkages are involved in the structural integrity of the envelope of EB. It appears that the liberation of the spherical envelope component is due to the disruption of certain of these ionic linkages, rather than to the activity of a lytic enzyme. The components which can be released by alkali treatment may be associated with the envelope in a manner similar to the ionic association of lipopolysaccharide in the cell envelopes of gram-negative bacteria (1, 21). ACKNOWLEDGMENTS The augmenting effect of EDTA may be due to We wish to excellent technical assistance acknowledge the deprivation of cations involved in such ionic of Elizabeth Brownridge andtheSamuel Garrison. bonding. Upon the addition of cations, reaggreThis work was supported by Public Health Service grant gation or reassembly of components liberated AI-00868 from the National Institute of Allergy and Infectious from the outer membrane of gram-negative Diseases. bacteria occurs (6, 10). Gilleland et al. (10) LITERATURE CITED demonstrated that Tris-EDTA removed spheri1. M. and R. G. Eagon. 1966. Role of multivalent Asbell, A., cal and rodlike particles which were located in cations in the organization, structure, and assembly of the inner layer of the outer membrane of Pseuthe cell wall of Pseudomonas aeruginosa. J. Bacteriol. domonas aeruginosa, and that on the addition 92:380-387.

EFFECT OF ALKALI ON C. PSITTACI

VOL. 125, 1976 2. Brown, A. D. 1964. Aspects of bacterial response to the ionic environment. Bacteriol. Rev. 28:296-329. 3. Brown, A. D. 1964. The development of halophilic properties in bacterial membranes by acylation. Biochim. Biophys. Acta 93:136-142. 4. Buckmire, F. L. A., and R. A. MacLeod. 1965. Nutrition and metabolism of marine bacteria. XIV. On the mechanism of lysis of a marine bacterium. Can. J. Microbiol. 11:677-691. 5. Costerton, J. W., J. M. Ingram, and K.-J. Cheng. 1974. Structure and function of the cell envelope of gram-negative bacteria. Bacteriol. Rev. 38:87-110. 6. DePamphilis, M. L. 1971. Dissociation and reassembly of Escherichia coli outer membrane and of lipopolysac7.

8.

9.

10.

11.

12.

13. 14.

15.

charide, and their reassembly onto flagellar basal bodies. J. Bacteriol. 105:1184-1199. Fan, D. P. 1970. Cell wall binding properties of the Bacillus subtilis autolysin(s). J. Bacteriol. 103:488-493. Freer, J. H., and M. R. J. Salton. 1971. The anatomy and chemistry of gram-negative cell envelopes, p. 67-126. In G. Weinbaum, S. Kadis, and S. J. Ajl (ed.), Microbial toxins, vol. IV, Bacterial endotoxins. Academic Press Inc., New York. Garrett, A. J., M. J. Harrison, and G. P. Manire. 1974. A search for the bacterial mucopeptide component, muramic acid, in Chlamydia. J. Gen. Microbiol. 80:315-318. Gilleland, H. E., J. D. Stinnett, I. L., Roth, and R. G. Eagon. 1973. Freeze-etch study of Pseudomonas aeruginosa: localization within the cell wall of an ethlenediaminetetraacetate-extractable component. J. Bacteriol. 113:417-432. Hoare, D. G., and D. E. Koshland. 1967. A method for the quantitative modification and estimation of carboxylic acid groups in proteins. J. Biol. Chem. 242:2447-2453. Koike, M., K. Iida, and T. Matsuo. 1969. Electron microscopic studies on the mode of action of polymyxin. J. Bacteriol. 97:448-452. Leive, L. 1965. Release of lipopolysaccharide by EDTA treatment of E. coli. Biochem. Biophys. Res. Commun. 21:290-296. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-296. Manire, G.OP., and A. Tamura. 1967. Preparation and chemical composition of the cell walls of mature

16

17.

18. 19.

20. 21.

22.

23. 24.

25.

26.

27.

28.

307

infectious dense forms of meningopneumonitis organisms. J. Bacteriol. 94:1178-1183. Matsumoto, A., N. Higashi, and A. Tamura. 1973. Electron microscope observations on the effects of polymyxin B sulfate on cell walls of Chlamydia psittaci. J. Bacteriol. 113:357-364. Matsumoto, A., and G. P. Manire. 1970. Electron microscopic observations on the fine structure of cell walls of Chiamydia psittaci. J. Bacteriol. 104:1332-1337. Newton, B. A. 1956. The properties and mode of action of the polymyxins. Bacteriol. Rev. 20:14-27. Repaske, R. 1958. Lysis of gram-negative organisms and the role of versene. Biochim. Biophys. Acta 30:225-232. Reynolds, E. S. 1963. The use of the lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17:208-212. Schnaitman, C. A. 1971. Effect of ethylenediaminetetraacetic acid, Triton X- 100, and lysozyme on the morphology and chemical composition of isolated cell walls of Escherichia coli. J. Bacteriol. 108:553-563. Smith, P. F., T. A. Langworthy, W. R. Mayberry, and A. E. Houghland. 1973. Characterization of the membrane of Thermoplasma acidophilum. J. Bacteriol. 116:1019-1028. Tamura, A., and N. Higashi. 1963. Purification and chemical composition of meningopneumonitis virus. Virology 20:596-604. Tamura, A., and G. P. Manire. 1967. Preparation and chemical composition of the cell membranes of developmental reticulate forms of meningopneumonitis organisms. J. Bacteriol. 94:1184-1188. Tamura, A., and G. P. Manire. 1968. Cytochrome C reductase activity of meningopneumonitis organisms at different stages of development. Proc. Soc. Exp. Biol. Med. 129:390-393. Tamura, A., A. Matsumoto, and N. Higashi. 1967. Purification and chemical composition of reticulate bodies of the meningopneumonitis organisms. J. Bacteriol. 93:2003-2008. Tamura, A., A. Matsumoto, G. P. Manire, and N. Higashi. 1971. Electron microscopic observations on the structure of the envelopes of mature elementary bodies and developmental reticulate forms of Chiamydia psittaci. J. Bacteriol. 105:355-360. Watson, M. C. 1968. Staining of tissue sections for electron microscopy with heavy metals. J. Biophys. Biochem. Cytol. 4:475-478.

Effect of alkali on the structure of cell envelopes of Chlamydia psittaci elementary bodies.

JOURNAL OF BACTERIOLOGY, Jan. 1976, p. 300-307 Copyright i 1976 American Society for Microbiology Vol. 125, No. 1 Printed in U.S.A. Effect of Alkali...
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