JOURNAL OF BACTERIOLOGY, Oct. 1967, p. 1178-1183Copyright i) 1967 American Society for Microbiology

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

Preparation and Chemical Composition of the CellWalls of Mature Infectious Dense Forms of

Meningopneumonitis OrganismsG. P. MANIRE AND AKIRA TAMURA

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

Received for publication 22 June 1967

Relatively large-scale production and purification of meningopneumonitis organ-isms was developed for chemical and immunological studies on cell walls of the in-fectious dense forms. By disruption of purified organisms with glass beads in aMickle shaker, highly purified preparations of cell walls were obtained by sucrosedensity gradient centrifugation, enzyme digestion, and sodium dodecyl sulfate treat-ment. The dry-weight recovery of purified cell walls from intact organisms was about13%. When32P-labeled preparations of cell wails were fractionated into acid-soluble,lipid, ribonucleic acid (RNA), deoxyribonucleic (DNA), and residual fractions,about 80% of the 32P in cell wall preparations was recovered in the phospholipidfraction, which corresponded to about 3% of the total phospholipid in the intactorganisms. About 7% of the 32P in purified cell walls was recovered in the RNA andDNA fractions respectively, but this corresponds to only about 0.4% of the 32Pfound in those fractions in intact organisms. From dry-weight determinations, itwas calculated that the purified cell wall preparations contained only 0.6% total nu-

cleic acids, and these are probably not true cell wall constituents. These cell wallscontained 70 to 75% protein, corresponding to about 14% of the protein in intactorganisms. Amino acid analysis of these proteins showed the existence of all commonamino acids, glucosamine, and galactosamine. However, no muramic acid was

detected by the methods employed.

In studies on the relationship of the psittacosisorganisms to bacteria, Jenkin (1) was able toproduce cell walls of the meningopneumonitis(MP) strain cultivated in the chick embryo bytreatment with sodium deoxycholate and trypsin.He reported the presence of 9 to 10 amino acids,hexosamine, reducing sugars, phospholipid, andtrace amounts of muramic acid in these prepara-tions. However, all suspensions of organisms usedfor these experiments contained mixtures of thedevelopmental reticulate forms and the matureinfectious dense forms, and no reports have beenpublished on chemical composition of the cellwalls of separate particles.

Following the development of methods for thepreparation of purified suspensions of the denseform of the MP organisms by Tamura andHigashi (8), Manire (3) reported on the prepara-tion of purified cell walls of dense forms by acombination of sonic treatment with glass beads,enzyme treatment, and sucrose density gradientcentrifugation. When finely shadowed fragments

were studied by electron microscopy, the innerlayer of the cell wall appeared to be composed ofregular geometric arrangements of hexagonallypacked macromolecules. This method has a def-inite disadvantage in that sonic treatment pro-duces excessive fragmentation of all componentsof the organism, with a consequent low yield ofcell walls.The recent development in this laboratory of

methods for relatively large-scale production ofthese organisms, and of improved methods for cellwall preparation, have made possible studies onthe chemical composition of purified cell walls ofthe dense forms of the organism. This paper is areport of these experiments.

MATERIALS AND METHODS

Organisms and cells. The Cal 10 strain of the MPorganism was propagated in L-cell suspension culture.The methods for propagation were essentially thesame as those described previously (8) except thatL cells were grown in 250-ml Erlenmeyer flasks

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VOL. 94, 1967 CELL WALLS OF MENINGOPNEUMONITIS ORGANISMS

containing 60 to 70 ml of medium and were incubatedat 37 C on an Eberback rotary shaker at 100 cyclesper min. By the use of two-deck shakers, as many as80 such cultures can be used in an experiment. Whenlarger amounts of cells were needed, 1- and 2-literspinner cultures were also prepared with the samemedium.

Purification of dense bodies. The method as pre-viously described was used for purification of sus-pensions of the infectious dense forms of MP (3, 8).Such preparations contained almost no contaminatingmaterials or reticulate bodies as seen by electronmicroscopy. In all experiments, these highly purifieddense bodies were used as starting materials forpreparation of cell walls.

Electron microscopy. The, methods of samplepreparation were previously reported (3). The speci-mens were shadowed with chromium or with plat-inum-palladium alloy and were examined in anAkashi Type TRC 50 electron microscope.

Density gradient centrifugation. Sucrose, potassiumtartrate, and cesium chloride density gradient col-umns were prepared in the nitro-cellulose centrifugetubes by use of a Buchler polystatic pump. Withsmall amounts of material, 0.5- to 1.0-ml amountsof cell wall suspension were layered on 4-ml gradientcolumns and centrifuged in the SW 39 rotor in aSpinco model L centrifuge. For large amounts ofmaterial, 5- to 8-ml amounts of the suspension werelayered on 20-ml gradient columns and centrifugedin an SW 25 rotor.

Experiments with 32p. The preparation of 32plabeled organisms and the methods for chemicalfractionation were described previously (8). 32P-labeled samples in solution were dried on aluminumor stainless-steel planchets and counted by use of aNuclear-Chicago gas-flow counter. No correction forself-absorption was necessary.

Analysis of protein content. Protein was routinelydetermined by the method of Lowry et al. (2) withcrystalline bovine serum albumin as the standard.This method is based on the content of phenylalanineand tyrosine in the samples, and the absolute contentof protein was determined by correcting for thecontent of these amino acids in the preparations.

Analysis of dry weight. For analysis of dry weightof the samples, the purified dense bodies or cell wallsin suspension were washed in distilled water byrepeated centrifugation. Each 1-ml portion of thesuspension was put in a small aluminum or glassdish and dried at 60 C for 3 to 5 hr, followed byvacuum desiccation over phosphorus pentoxide for1 week, by which time the sample had reached con-stant weight.

Amino acid analysis. Amounts of 5 to 20 mg ofpurified dense bodies or cell walls were hydrolyzedat 100 C with 3 ml of 4 N HC for 10 hr or 6 N HCIfor 18 hr in a sealed glass tube. The hydrolysates weredried in a rotary evaporator. The amino acid analyseswere performed by use of a Spinco Beckman type120 B automatic amino acid analyzer.

Materials. Carrier-free nP-orthophosphate waspurchased from Oak Ridge National Laboratory.The trypsin used was twice crystallized salt-free

trypsin purchased from Nutritional BiochemicalsCorp., Cleveland, Ohio. Crystalline ribonuclease anddeoxyribonuclease were also obtained from Nutri-tional Biochemicals Corp. CsCl2 was obtained fromGallard Schlesinger Chemical Mfg. Corp., CharlesPlace, N.Y. Potassium tartrate was purchased fromJ. T. Baker Chemical Co., Phillipsburg, N.J., and wasused without further purification. Muramic acid waspurchased from Sigma Chemical Co., St. Louis, Mo.

RESULTSPreparation of cell walls. We previously de-

scribed a method for the disruption of psittacosisorganisms by sonic treatment in the presence ofglass beads in which the cell walls were brokendown to various-sized fragments (3). In pre-liminary experiments, it was subsequently foundthat treatment of these organisms in a Mickledisintegrator by the following procedures resultedin disruption of the cell walls with a minimum offragmentation. Suspensions (4 ml) of purifieddense bodies (25 to 500 mg) in 0.033 Mtris(hydroxymethyl)aminomethane (Tris) buffer(pH 7.4) were mixed with 4 g of glass beads (no.18), and homogenized by use of a Mickle appara-tus (60 cycle/sec) at 5 C for 5 min. In preliminarytests to determine the optimal period for homog-enization, many organisms were disrupted after2 min, almost all particles were disrupted in 5 min,and only small fragments remained after 15 minof treatment. Figure 1 shows an electron micro-graph of a homogenate after 5 min of treatment.

After homogenization, treated suspensions wereallowed to stand for a few minutes and the super-natant fluids were separated by capillary pipette.The glass beads were washed six times with 1 mlof 0.033 M Tris buffer (pH 7.4). The fluid of thesixth washing showed almost no turbidity. Thesupernatant fraction and the washing fluids werepooled, and 5 to 7 ml of this homogenate waslayered on 20-ml density gradient columns of 5 to45% sucrose and centrifuged at 8,000 rev/min for30 min in the SW25 rotor of a Spinco centrifuge.The cell walls were found in a band in the middleof the column. Occasionally another small opaqueband was observed near the bottom ofthe column;this band was found to be composed of intactdense forms of MP. Figure 2 shows an electronmicrograph made from the content of such asucrose density gradient band.The cell wall bands in the sucrose density

gradient columns were harvested by capillarypipette, diluted three- to fivefold with distilledwater, and centrifuged at 10,000 X g for 1 hr.The precipitate obtained was suspended in 2 to 10ml of 0.2 M Tris buffer (pH 7.4) containing 0.02M MgCl2, and was sonic-treated for 1 min to makea homogeneous suspension. This suspension wasincubated with 0.1 mg each of ribonuclease and

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MANIRE AND TAMURA J. BACTERIOL.

FIG. 1. Electron micrograph ofthe homogenate ofdense bodies after 5-min treatment by Mickle shaker with glassbeads. X 16,000.

FIG. 2. Electron micrograph of partially purified cell walls obtained after sucrose density gradient centrifuga-tion. X 32,000.

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CELL WALLS OF MENINGOPNEUMONITIS ORGANISMS

deoxyribonuclease per ml at 37 C for 2 hr. Afterincubation, 1 mg of trypsin per ml was added, andthe suspension was further incubated for 2 hr.The suspension of cell walls was then dilutedthreefold with 0.2 M Tris buffer (pH 7.4) andcentrifuged at 8,000 X g for 1 hr. This enzymetreatment removed about 80% of ribonucleic acid(RNA) and 70% of deoxyribonucleic acid (DNA)present in the cell walls before treatment, but nomorphological changes were seen by electronmicroscopy.The cell wall pellet was suspended in 4 to 10 ml

of water, and 1% sodium dodecyl sulfate (SDS)was added dropwise with vigorous shaking untilthe final concentration of SDS was 0.5%. Thesuspension was incubated at 37 C for 2 hr and wasthen centrifuged at 10,000 x g for 1 hr. The SDStreatment decreased the turbidity of the suspen-sion by about one-half. The precipitate was sus-pended in distilled water and again centrifuged at10,000 x g for 1 hr to remove the SDS. Thepellet was finally suspended in distilled water.Figure 3 shows an electron micrograph of puri-fied cell walls obtained by the above procedure.

Chemical composition of purified cell walls.Purified 32P-labeled cell walls of infectious densebodies were prepared as above, and the 32p distri-bution was analyzed by Schmidt-Thannhauserfractionation. The protein contents of the original

dense body suspension and of the cell wall suspen-sions obtained by sucrose density gradient andafter further treatment with SDS were determined.The results of two experiments are shown in Table1. About 75 to 80% of 32P in purified cell walls wasrecovered in the lipid fraction, and the residual32P was distributed in other fractions. The phos-pholipids extracted from the cell walls containedabout 3% of the 32p of the intact organisms.The recovery in purified cell walls of dry weight

and of protein from intact dense bodies wasmeasured in four experiments (Table 2). The re-covery as measured by dry weight averaged 12.7%and the recovery of protein averaged 14.2% ofthat in intact dense bodies.

Direct estimation of the nucleic acid content ofcell wall preparations could not be made becausethe amounts were too low for chemical determina-tion. However, it is possible to estimate thesefrom the 32p recovery data shown in Table 1 andfrom direct analyses of whole dense bodies.These estimates are shown in Table 3. In column 1of Table 3, the average results of three analyses forprotein, RNA, and DNA of whole dense bodiesare shown, as previously reported (7). The lipidcontent of these intact organisms was estimatedto be < 20% by subtraction of combined protein,RNA, and DNA from total. No carbohydrate isaccounted for in these calculations.

FIG. 3. Electron micrograph ofpurified cell walls ofdense bodies. X 32,000.

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MANIRE AND TAMURA

TABLE 1. Distribution anid recovery of 32p invarious fractions of labeled dense bodies anid

ofpurified cell walls ofMP organisms

Dense bodies Purified cell wallsRecovery

Expt Fraction in cellCounts/ Distri- Counts/ Distri- wallsmin bution min bution

(Xl,000) (%) (X1,000) (%) %

1 Lipid 1,210 37.4 34.4 80.2 2.8RNA 917 28.4 2.8 6.5 0.3DNA 834 25.8 2.8 6.5 0.3Total 3,230 42.9

2 Lipid 3,150 40.2 107.0 75.7 3.4RNA 2,060 26.3 9.7 6.8 0.5DNA 1,990 25.4 11.0 7.8 0.6Total 7,840 141.2

TABLE 2. Recovery of dry weight and protein incell walls of dense bodies ofMP organism

Dense bodies Cell wallsRecovery

Expt in cellDry wt Protein Dry wt Protein walls (%)(mg) (mg) (mg) (mg)

1 20.3 2.1 10.22 72.5 11.0 15.23 24.8 3.1 12.54 34.2 5.4 15.8

TABLE 3. Comparison of chemical compositioni ofintact dense bodies and purified cell walls

of meninigopneumonitis organiisms

Amt containedAmt contained in purified cell Total

Fraction in 100 mg of walls derived content inwhole dense from 100 mg of purified cellbodies (mg) whole dense walls (%)

bodies (mg)

Dry weight lOOa 12.7c 100Protein ....... 64.4a 9. 2c 72RNA ........ 8. Oa 0. 03d 0.25DNA.7.6a 0.04d 0.29Lipid. <20b <o0.62d <5.1

a Data taken from reference 7.b Estimated by subtraction of protein, RNA,

and DNA content from dry weight.c Direct analyses on purified cell walls.d Estimates obtained by multiplying (milli-

grams of content in whole dense bodies) X (aver-age per cent recovery in cell walls from Table 1).

In the second column of Table 3, the estimatedcontents of cell walls are presented. The values fordry weight and protein content were obtained bydirect measurement. The values for RNA, DNA,and lipid content were calculated from column 1,

and the total 32p recovery data were obtained asshown in Table 1.From these calculations, the cell walls appear

to be composed largely of protein; combinedRNA and DNA comprise less than 0.6% of thedry weight of cell walls and probably are not truecell wall constituents. Although about 3% of thephospholipid of the whole organism is recoveredin the cell walls, the total lipid content is less than5%, dry weight.

Similar preparations of whole organisms and ofpurified SDS-treated cell walls were hydrolyzedand analyzed for amino acid content. The resultsof two experiments are shown in Table 4 wherethe amino acid content of cell walls of Escherichiacoli (5) and group A streptococci (10) are pre-sented for comparison. The intact organisms andthe purified cell walls contain all the commonnaturally occurring amino acids. Relatively largeamounts of alanine, aspartic acid, glutamic acid,glycine, and serine were found in these purifiedcell walls. This distribution of amino acids issimilar to that in the gram-negative bacteria suchas E. coli.

In the analyses of amino acid composition doneto date, small amounts of glucosamine and galac-tosamine have been found in these dense-body

TABLE 4. Aminio acid compositions of MP deniseforms anzd their cell walls, anid comparisonwith those of cell walls of Streptococcus

group A and Escherichia colia

Amino acids

Alanine ..Arginine......Aspartic acid.Glutamic

acid .......Glycine.......Half cystine.Histidine.....Isoleucine ....Leucine ......Lysine ....Methionine ...Phenylala-

nine........Proline ....Serine ....Threonine.Tyrosine......'Valine.... ...

MP denseforms

9.64.1

10.1

9.48.91.61.85.08.07.91.8

3.85.28.06.62.65.2

Cell Cell wallswalls of of Strep-MP dense tococcusforms group Ab

9.9 57.53.7 -

12.2 2.9

8.7 15.29.1 2.14.1 -

1.3 -3.6 0.96.4 1.45.4 15.01.0 -

3.8 0.86.69.4 1.77.6 1.73.0 0.84.2 -

Cell wallsof E. coli'

13.14.611.1

9.88.6

1.25.98.55.71.0

3.82.77.46.73.86.1

a The results are shown as micromoles per centof total amino acids.

b Data from Tepper, Hayashi, and Barkulis (10).c Data from Salton (5).

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VOL. 94, 1967 CELL WALLS OF MENINGOPNEUMONITIS ORGANISMS

cell wall preparations. However, no diaminopi-melic acid and no muramic acid have beendetected in either intact dense bodies or their cellenvelopes.When purified muramic acid was mixed with

an acid hydrolysate of crystalline bovine serumalbumin, the minimal amount of muramic acidwhich could be detected on the chromatogram ofthe amino acid analyzer was 20 ,ug. In the analysesof amino acid content of dense bodies and theircell walls, 10-mg amounts of purified preparationswere hydrolyzed and analyzed. It may thereforebe concluded that the muramic acid content indense bodies and cell walls is less than 0.2%.

DISCUSSIONFurther knowledge of the nature of the mature

infectious dense forms of psittacosis organismsand of their cell walls has been provided by thesestudies. Such organisms are surrounded by a rigidcell wall which is resistant to physical methods ofdisruption, but which can be broken by sonictreatment with glass beads or by treatment in aMickle disintegrator with glass beads. The latterapparatus appears to open the cell wall withoutexcessive fragmentation.By the use of sucrose density gradient centrif-

ugation, followed by treatment with enzymes andSDS, very clean preparations of the cell walls maybe obtained. Evidence for the purity of these prep-arations was provided by electron microscopy andby chemical analyses. Approximately 13% (dryweight) of the intact organisms was recovered inthe final cell wall fraction.The purified cell walls contain phospholipids,

as 70 to 80%C of 32p in purified cell wall was foundin the lipid extracts. The 3P content in the lipidfraction of cell walls corresponds to about 3%of the total 32p incorporated in the lipid fractionof the whole organism. About 7% of 32p in puri-fied cell walls was recovered in RNA and DNArespectively, but this corresponds to only about0.4% of the total 32p incorporated into RNA andDNA iractions of intact organisms, and the con-tent of RNA and DNA in cell walls was calculatedto be less than 0.3%.

Similarity of the amino acid composition ofthese cell walls to those of gram-negative bacteriawas found. However, muramic acid was not de-tected in acid hydrolysates of cell walls duringamino acid analysis, although muramic acid inpsittacosis organisms has been reported (1, 4).

The implications of these findings relative tothe developmental cycle of psittacosis organismsis discussed in an accompanying paper, in whichsimilar studies on the cell membranes of the largedevelopmental noninfectious reticulate forms (9)are reported.

ACKNOWLEDGMENTS

We wish to express appreciation to Martin Griffenfor technical assistance and to Lawrence A. Wilsonwho 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 CITED

1. JENKIN, H. M. 1960. Preparation and properties ofcell walls of the agent of meningopneumonitis.J. Bacteriol. 80:639-647.

2. LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR,AND R. J. RANDALL. 1951. Protein measure-ment with the Folin phenol reagent. J. Biol.Chem. 193:265-275.

3. MANIRE, G. P. 1966. Structure of purified cellwalls of dense forms of meningopneumonitisorganisms. J. Bacteriol. 91:409-413.

4. PERKINS, H. R., AND A. C. ALLISON. 1963. Cellwall constituents of rickettsia and psittacosislymphogranuloma organisms. J. Gen. Mi-crobiol. 30:469-480.

5. SALTON, M. R. 1964. The bacterial cell wall.Elsevier Publishing Co., New York.

6. SCHMIDT, G., AND S. J. THANNHAUSER. 1945. Amethod for the determination of deoxyribo-nucleic acid, ribonucleic acid and phospho-protein in animal tissues. J. Biol. Chem.161:83-89.

7. TAMURA, A. 1964. Biochemical studies on themechanism of infection of meningopneumonitisagent. Ann. Rept. Inst. Virus Res. KyotoUniv. 7:1-13.

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

9. TAMURA, A., AND G. P. MANIRE. 1967. Preparationand chemical composition of the cell mem-branes of developmental reticulate forms ofmeningopneumonitis organisms. J. Bacteriol.94:1184-1188.

10. TEPPER, B. S., J. A. HAYASHI, AND S. S. BARKULIS.1959. Studies of streptococcal cell walls. V.Amino acid composition of cell walls ofavirulent group A hemolytic streptococci.J. Bacteriol. 79:33-38.

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