JOURNAL OF BACRIOLOGY, Oct. 1967, p. 1184-1188Copyright 0 1967 American Society for Microbiology

Vol. 94, No. 4Printed In U.S.A.

Preparation and Chemical Composition of the CellMembranes of Developmental Reticulate Forms

of Meningopneumonitis OrganismsAKIRA TAMURA AND G. P. MANIRE

Department of Bacteriology and Immunology, School of Medicine, University of North Carolina, Chapel Hill,North Carolina 27514

Received for publication 22 June 1967

The outer limiting membranes of developmental reticulate forms of the meningo-pneumonitis organism were purified by a combination of differential centrifugation,trypsin digestion, and sodium dodecyl sulfate treatment, and their physical andchemical properties were compared with those of outer envelopes of mature denseforms of this organism. Reticulate bodies were easily disrupted by short periods ofsonic treatment and were lysed by trysin digestion, in contrast to the dense bodieswhich were resistant to these treatments. In electron micrographs, reticulate bodymembranes were seen as very thin, flattened structures, whereas dense-body enve-lopes showed folding rigid membranes. The results of chemical fractionation of 32plabeled purified preparations indicated that reticulate body membranes have smalleramounts of phospholipid, and are more dense than cell walls of the mature forms.The analysis of amino acid composition of reticulate body cell membranes showedthat they do not contain cystine or methionine, both of which were found in cellwalls of dense bodies. These results clearly show that there are significant differencesin the chemical and physical properties of the outer envelopes of the developmentaland mature forms of this organism.

That psittacosis organisms undergo a complexdevelopmental cycle after entry into susceptiblecells has been clearly demonstrated by electronmicroscopic studies (1) and by the development ofmethods for the separate preparation of highlypurified suspensions of the infectious rigid denseforms (5) and of the early noninfectious fragilereticulate forms (6) of these organisms. It wouldthus appear that a comparative study of thechemical composition and other characteristics ofboth reticulate and dense forms would be valuablein the study of the multiplication cycle of theseorganisms. One such approach has been to isolatethe membranes or walls of both forms separately,and to compare their natures. The results ob-tained in studies on the cell walls of the denseform are presented in an accompanying paper(3). This report concerns the preparation of puri-fied suspensions of membranes from suspensionsof reticulate bodies of meningopneumonitis (MP)organisms together with studies on their chemicalcomposition and physical characteristics.

MATERIALS AND METHODSMethods. The methods used for L-cell propagation,

electron microscopy, density gradient centrifugation,

isotope labeling, and chemical analysis, and thesource of L cells and MP organisms, were the same asin the preceding report (3).

Purified suspensions of reticulate bodies wereprepared essentially as described by Tamura et al. (6).

Materials. Trypsin was purchased from GeneralBiochemicals, Chagrin Falls, Ohio. Crystallinepapain and lysozyme were purchased from NutritionalBiochemicals Corp., Cleveland, Ohio.

RESULTS

Effect of sonic treatment on reticulate bodies. Apurified suspension of reticulate bodies in 5 ml ofphosphate-buffered saline (PBS), was treated in a9-kc Raytheon sonic oscillator at maximal platevoltage and frequency. Decreases in turbidity wererecorded in a spectrophotometer at a wavelengthof 550 m,. As shown in Fig. 1, the turbidity of thesuspension of reticulate bodies decreased to lessthan half its original level after 2-min treatment,in contrast to that of dense bodies, which showedonly a 30% decrease after 10 min of sonic-treat-ment. By electron microscopy, it was observedthat few intact reticulate bodies remained after30 sec of sonic treatment, and in 5 min all thereticulate bodies were completely fragmented.

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VOL. 94, 1967 CELL MEMBRANES OF MENINGOPNEUMONITIS ORGANISMS

100

80

601

DENSE BODIES

RETICULATE BODIES

401

20c

2 3 4 5 6 7 8 9 10

MINUTES OF SONICATIONFig. 1. Decrease in turbidity ofpurified suspensions

of dense bodies and of reticulate bodies after sonictreatment.

Efforts to purify the membranes of reticulatebodies from sonically disrupted organisms bydifferential and density gradient centrifugationwere unsuccessful, owing to the extreme variationin fragment size.

Effect of enzymes and chemical treatment onreticulate bodies. The effects of various enzymesand chemicals on suspensions of reticulate bodieswere studied. Suspensions of purified reticulatebodies in 0.1 M tris(hydroxymethyl)amino-methane (Tris) buffer (pH 8.5) were incubated at37 C alone and with each of the following at thefinal concentration shown in parentheses: lyso-zyme (100 ,ug/ml), ethylendiaminetetraacetate(0.005 M), papain (100 Ag/ml), trypsin (200,ug/ml), mercaptoethanol (2%), and sodium laurylsulfate (SDS; 0.25%.) The turbidity of each sus-pension was determined at 30 sec, at 1, 2, 3, and60 min, and at 24 hr (Fig. 2). Lysozyme had noeffect on reticulate bodies, although their fragilitywas evident in that both control and lysozyme-treated suspensions showed a constant decrease inturbidity. Mercaptoethanol- and papain-treatedcells showed a slow loss of turbidity, whereas bothSDS and trypsin caused marked reduction inturbidity within 1 to 4 min. When treated cellswere examined in shadow-cast preparations byelectron microscopy, it was seen that trypsin,SDS, and mercaptoethanol were effective in re-moving most of the internal constitutents of thereticulate bodies.

Purification of cell membranes of reticulatebodies. On the basis of the above observation, itwas possible to prepare highly purified suspen-

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580 X amercoptoethonol

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20-SD'S

2 3 4 60 24 hoursMINUTES OF TREATMENT

FIG. 2. Decrease in turbidity of the purified sus-pensions of reticulate bodies after incubation at 37 Cwith enzymes, SDS, and mercaptoethanol.

sions of reticulate body membranes by the follow-ing procedure.

Purified reticulate bodies were suspended in0.1 M Tris buffer (pH 8.5) and incubated withtrypsin (200 Ag/ml) at 37 C for 30 min and thenat 2 to 4 C overnight. After centrifugation at800 X g for 10 min, the resulting supernatantfluid was centrifuged at 8,000 X g for 30 min, andthe precipitate obtained was suspended in a smallamount of distilled water.Equal parts of 0.2 M Tris buffer (pH 7.4) con-

taining 0.02 M MgCl2 and 0.1 volume of ribo-nuclease and deoxyribonuclease solution (100,ug/ml each, final concentration) were added tothe suspension and incubated at 37 C for 2 hr.After incubation, the mixture was again centri-fuged at 8,000 X g for 30 min and the precipitatewas suspended in distilled water. This suspensionwas mixed with an equal amount of 0.5% SDSand incubated at 37 C for 2 hr. The suspensionwas then centrifuged at 800 X g for 10 min, andthe cell membranes were then centrifuged at8,000 X g for 30 min, washed once by repeatingthe centrifugation cycle, and suspended in distilledwater.The appearance of the membranes after treat-

ment with trypsin and the final preparation ofmembranes are shown in Fig. 3 and 4. The highdegree of purity of these membranes and thestepwise removal of the internal constituents isevident from these micrographs. These mem-branes are much flatter and show little evidence ofthe characteristic rigidity of the dense-body cellwalls, which show folding figures as seen in thepreceding paper (3).

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FIG. 3. Electron micrograph of partially purified reticulate body membranes obtained after treatment withtrypsin. X 24,000.

FIG. 4. Electron micrograph offinially purified reticulate body membranes obtained after treatment with SDS. X24,000.

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VOL. 94, 1967 CELL MEMBRANES OF MENINGOPNEUMONITIS ORGANISMS

TABLE 1. Chemical distribution of 32p in purified reticulate bodies and their cell membranes"

Wholereticulatbodi Cell membranes after Cell membranes afterWhole reticulate bodies trypsin treatment SDS treatment

Fraction Recovery (%)

CPM (X 1,000) Distribution CPM (X Distribution CPM (X Distribution(%) 1,000) (%) 1,000) (%)

Acid soluble 306 19.5 59.7 17.8 15.2 66.7 4.9Lipid ............ 487 31.1 146 43.6 3.37 14.8 0.7RNA ............ 607 38.8 115 34.4 2.11 9.2 0.3DNA ............ 105 9.6 8.3 2.5 1.38 6.0 1.3Residue.......... 15.3 1.0 5.9 1.8 0.70 3.1 4.6

Total ............ 1,520 334.9 22.76 1.5

a The protein content (and the per cent recovery of protein) in the various cell preparations was asfollows: whole reticulate bodies, 10.2 mg; cell membranes after trypsin treatment, 1.79 mg (17.5%);cell membranes after SDS treatment, 0.507 mg (4.95%).

Chemical composition of purified membranes.Reticulate-body cell membranes were prepared asabove from 32P-labeled organisms and frac-tionated by the Schmidt-Thannhauser technique.The distribution of 32p in the acid-soluble, lipid,RNA, DNA, and residue fractions of wholereticulate bodies and of cell membranes aftertrypsin treatment and after trypsin plus SDStreatment as above are shown in Table 1. Trypsintreatment resulted in removal of 70% of the lipid,80% of the RNA, and 90% of the DNA, andfurther treatment with ribonuclease, dexoyribo-nuclease, and SDS resulted in removal of 98% oflipid, 98% of RNA, and 83% of DNA whichremained in the trypsin-treated membranes. Thefinding that the lipid fraction contained only14.8% of the total 32P in the purified reticulate-body membranes, which represents only 0.7% of32P-labeled phospholipid of intact reticulatebodies, indicates a low content of phospholipidsin these membranes. The recovery of proteinrepresented about 5% of that in the intact reticu-late bodies.

Approximately 5 mg of purified reticulatebodies and a similar amount of purified mem-branes were hydrolyzed in 3 ml of 4 N HCl at 100C for 10 hr, or 6 N HCl at 120 C for 18 hr, and theamino acid composition was determined as in thepreceding paper (3). These analyses are shown inTable 2. There are few differences in the aminoacid compositions of intact reticulate bodies, cellmembranes of reticulate bodies, and cell walls ofdense bodies, except for the absence of bothmethionine and cystine in the reticulate-bodymembranes.No evidence of muramic acid was found in this

analysis.Density of cell membranes. Purified suspensions

of reticulate-body cell membranes were layeredon top of a 20 to 60%o CsCl2 density gradient

TABLE 2. Amino acid compositions of reticulatebodies and their cell membranesa

Intact Cell membranesAmino acids reticulate of reticulate

bodies bodies

Alanine. 9.8 11.0Arginine.4.6 3.0Aspartic acid.10.9 12.1Glutamic acid.12.1 8.9Glycine.9.1 9.6Half cystine.1.0 0Histidine. 2.0 1.2Isoleucine.2.9 5.4Leucine. 8.7 7.9Lysine.6.6 4.8Methionine.2.1 0Phenylalanine.3.8 4.2Proline.5.9 6.4Serine. 8.0 9.7Threonine.5.4 7.9Tyrosine. 3.2 2.7Valine.4.1 5.3

a The data are shownof total amino acids.

as micromoles per cent

column and centrifuged at 20,000 rev/min for 60min in a Spinco SW 39 rotor. After centrifugation,the cell membranes of reticulate bodies formed asharp agglutinating band at the middle of thedensity column. Purified cell walls of dense bodieswere prepared and centrifuged under the sameconditions. These cell walls formed a band nearthe top of the column. This result shows that thedensity of cell walls of dense bodies was less thanthat of cell membranes of reticulate bodies. Por-tions of both bands were carefully removed bycapillary pipette, and the CsCl2 concentration inthe suspensions was analyzed with a Carl Zeissmodel A refractometer. The buoyant densities ofthe reticulate-body membranes and the dense-

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TAMURA AND MANIRE

body cell walls were calculated at 1.395 and 1.295,respectively.

DISCUSSION

We have succeeded in preparing highly purifiedsuspensions of the outer envelopes of the maturedense forms of MP organisms as reported in theprevious paper (3) and of the developmentalreticulate forms in sufficient quantity for bothmorphological and chemical studies, and havefound several distinct differences in these struc-tures.The reticulate bodies have very fragile, non-

rigid outer membranes; they are almost com-pletely destroyed by short-time sonic treatmentand are subject to considerable lysis on standing.In electron micrograph preparations, the isolatedpurified membranes of reticulate bodies areusually seen as very thin, completely flattenedstructures. In contrast, the dense bodies have veryrigid and resistant envelopes which resemble truebacterial cell walls (2, 3). These walls are highlyresistant to sonic treatment and other physicalmethods of cell disruption, and show foldingfigures in electron micrographs.The reticulate body membranes are readily

permeable both to trypsin and to the internalconstituents of the organism after trypsin treat-ment. It is not known whether this loss of internalmaterials is due to the effects of trypsin on themembrane or to digestion of the internal struc-tures. No effect of trypsin on the structure ofdense bodies can be detected and this enzyme isof value in their purification.Whereas 80% of the 32P in cell walls of the

dense form is found in the phospholipid fraction,representing about 3% of the phospholipid of thewhole organism (3), only 14% of the 32p in thereticulate-body membranes is in the phospholipidfraction, representing only 0.7% of the phospho-lipid of intact reticulate bodies. These results indi-cate that the phospholipid content in reticulate-body cell membranes is much less than that ofdense-body envelopes. The cell walls of densebodies have lower density than those of reticulate-body cell membranes. This may coincide with theobservations which show the existence of morelipid in the former.

Although cystine and methionine are bothfound in the cell walls of the dense forms, these

sulfur-containing amino acids are completelyabsent in reticulate-body membranes. This isbeing studied further to determine whether S-Sbonding may play a role in the formation of rigidmembranes of the mature dense forms.The findings described above represent pro-

found and significant differences in the nature ofthe outer envelopes of the developmental and themature forms of the psittacosis organisms. Thebiological significance of these differences, andthe mechanism of change from one rigid cell wallof the infectious dense form within a susceptiblecell to the fragile membrane of the reproductiveform, and again to the rigid membrane at the timeof maturation, are not known. One can speculatethat the reticulate body membrane is similar tothe cytoplasmic membrane of bacteria, and thishighly permeable structure is necessary for growthof this parasitic organism in a susceptible cell.

ACKNOWLEDGMENTSWe express appreciation to Marian Griffen for

excellent technical assistance, and to Lawrence A.Wilson, who made the electron micrographs.

This investigation was supported by Public HealthService grant AI-00868 from the National Institute ofAllergy and Infectious Diseases, and by PublicHealth Service General Research Support Award5 SOI-FR-05406.

LITERATURE CITED1. HIGASHI, N. 1965. Electron microscopic studies on

on the mode of reproduction of trachoma virusand psittacosis virus in cell culture. Exptl. Mol.Pathol. 4:24-39.

2. MANIRE, G. P. 1966. Structure of purified cell wallsof dense forms of meningopneumonitis or-ganisms. J. Bacteriol. 91:409-413.

3. MANIRE, G. P., AND A. TAMURA. 1967. Preparationand chemical composition of the cell walls ofmature infectious dense forms of m-ningo-pneumonitis organisms. J. Bacteriol. 94:1178-1183.

4. TAMURA, A. 1967. Isolation of ribosome particlesfrom meningopneumonitis organisms. J. Bac-teriol. 93:2009-2016.

5. TAMURA, A., AND N. HIHASHI. 1963. Purificationand chemical composition of meningopneumo-nitis virus. Virology 20:596-604.

6. TAMURA, A., A. MATSUMOTO, AND N. HIGASHI.1967. Purification and chemical composition ofreticulate bodies of the meningopneumonitisorganisms. J. Bacteriol. 93:2003-2008.

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