developmental cycle coxiella burnetii: structure and ... · carries out its development cycle in...

Vol. 147, No. 3JOURNAL OF BACTERIOLOGY, Sept. 1981, p. 1063-10760021-9193/81/091063-14$02.00/0

Developmental Cycle of Coxiella burnetii: Structure andMorphogenesis of Vegetative and Sporogenic Differentiations

THOMAS F. McCAUL AND JIM C. WILLIAMS*Department ofHealth and Human Services, National Institute ofAllergy and Infectious Diseases,

Laboratory ofMicrobial Structure and Function, Rocky Mountain Laboratories, Hamilton, Montana 59840

Received 8 January 1981/Accepted 26 May 1981

Coxiella burnetii is a gram-variable obligate intracellular bacterium whichcarries out its development cycle in the phagolysosome of eucaryotic cells.Ultrastructural analysis of C. burnetii, in situ and after Renografin purification,by transmission electron microscopy of lead-stained thin sections has revealedextreme pleomorphism as demonstrated by two morphological cell types, a largecell variant (LCV) and a small cell variant (SCV). Potassium permanganatestaining of purified rickettsiae revealed a number of differences in the internalstructures of the cell variants. (i) The outer membrane of the SCV and LCV werecomparable; however, the underlying dense layer of the SCV was much widerand more prominent than that of the LCV. The periplasmic space of the SCVwas not readily visualized, whereas the periplasmic space ofthe LCV was apparentand resembled that of other gram-negative bacteria. (ii) Complex internal mem-branous intrusions which appeared to originate from the cytoplasmic membranewere observed in the SCV. The LCV did not harbor an extensive membranoussystem. (iii) Some LCVs contained a dense body in the periplasmic space. Thisendogenous structure appeared to arise in one pole of the LCV as an electron-dense "cap" formation with the progressive development of a dense body approx-imately 130 to 170 nm in diameter which was eventually surrounded by a coat ofat least four layers. Our observations suggest that the morphogenesis of C.burnetii is comparable, although not identical, to cellular differentiation ofendospore formation. A developmental cycle consisting of vegetative and sporo-genic differentiation is proposed.

Coxiella burnetii, the etiological agent of Qfever, differs from other members of the familyRickettsiaceae in its high degree of resistance tophysical and chemical agents (4), unique phasevariation (55), variable Gram stain reaction (21),and DNA base composition (53, 64). C. burnetiihas been shown to be highly pleomorphic duringmultiplication within phagolysosomes of hostcells (7). The pleomorphic nature of C. burnetiiwas first described by Davis and Cox (15), whoobserved by light microscopy that minute coc-coid and granular forms, as well as bacillaryforms, of C. burnetii were present in cells ofinfected guinea pigs and in tissue cultures. Thesize of C. burnetii was so variable that someforms passed through filters. Ultrastructuralanalysis of C. burnetii, studied in chicken em-bryonic yolk sacs (YS) (1), tissue culture celllines (51), infected animal tissues (2, 26), andinfected peritoneal macrophages (31, 32) re-vealed distinct cell variants with marked mor-phological differences.

Cell variants of C. burnetii could be separatedinto two distinct zones in CsCl, sucrose, or iso-

pycnic Renografin gradients (9, 61). Althoughthe relationship between the cell variants of C.burnetii has not been fully elucidated, Wiebe etal. (61) speculated that they represented twostages in a complex developmental cell cyclesimilar to the one of Chlamydiapsittaci, anotherorder of obligate intracellular parasites. An al-ternative mechanism for the generation of cellvariants was suggested by Wiebe et al. (61) toinclude partial degradation of the small cell var-iants (SCVs) by lysosomal enzymes of host cellorigin, thereby rendering them large cell variants(LCVs). Thus, the SCV was considered a "nor-mal" nondegenerated cell apparently unaffectedby enzymatic processes. Physical stresses asso-ciated with storage of infected embryonic YSand purification of samples were also thought toplay contributing factors in generating LCVs(58).In contrast to the observations implicating

transverse binary fission (61) as the mode ofreplication of C. burnetii, a quite different intra-cellular behavior was proposed by Kordova etal. (33-36, 51). The multiplication process of C.

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1064 McCAUL AND WILLIAMS

burnetii was thought to be similar to that ofviral replication during the early phase of theinfection cycle. Evidence for this interpretationwas the demonstration of infectious, round par-ticles of C. burnetii, which passed through 40-nm collodion membranes (33). Such ultrafil-trates were capable of causing infection in YStissue culture cells (36). During this infectioncycle, fine granules appeared, and as the infec-tion proceeded, the vacuolized cytoplasm wasfilled with antigenic material of differing sizeand form. Subsequently, C. burnetii particlesappeared in the cytoplasm as masses of recog-nizable rickettsiae. Most authors (46, 59, 63)have, however, expressed some doubts as to thevalidity of these observations, ascertaining thattranverse binary fission is the customary andonly mode of replication.The purpose of this study is to resolve the

apparent discrepancy in our understanding ofthe relationship between the distinct cell var-iants and the developmental cycle of C. burnetii.Ultrastructural analysis, using several condi-tions of preparation, was therefore carried outon C. burnetii in situ in YS tissues and on viableRenografin-purified cells. The results indicatethat C. burnetii has certain features comparableto those of bacteria which undergo differentia-tion leading to endospore formation.

MATERIALS AND METHODSBacteria. C. burnetii strains employed in this study

were in various passage levels in guinea pigs, chickenembryo fibroblast tissue culture, and chicken embryoYS. The Ohio strain (5EP/2GP/2EP) was in phase Iwithout detectable phase II antigens, whereas theNine Mile strains were in phase I (307GP/1TC/1EP,clone 7) and phase II (90EP/lTC/4EP, clone 4), here-after designated CBOI, CB9MI, and CB9MII, respec-tively.

Preparation ofrickettsiae. C. burnetii were prop-agated in specific. pathogen-free type IV, antibiotic-free, fertile hen egg YS (H & N Hatchery, Redmond,Wash.). Stock cultures of C. burnetii were maintainedas 50% (wt/vol; grams of YS per milliliter of brainheart infusion broth [Difco Laboratories, Detroit,Mich.]) YS suspensions at-70°C. Stock cultures wereprepared by injecting C. burnetii into the YS ofchicken embryos on the 5th day postincubation. In-cubation was carried out at 350C in a humidity-con-trolled Jamesway incubator. On the 7th day postinfec-tion, the YS from live embryos were harvested, and a50% YS suspension in brain heart infusion was pre-pared by blending (Waring blender, model F.C. 114)for 30 s at a powerstat setting of 100. Stock cultureswere shell-frozen as 2-ml aliquots in sealed glass am-poules. Plaque-forming units inoculated into each YSwere 1.1 x 109 for CBOI, 1.5 x 107 for CB9MI, and 5.6X 107 for CB9MII. Mean survival time of the infectedembryos was 7.4 ± 0.2 days at 350C. Cultures destinedfor the separation of C. burnetii from host materialwere frozen in batches of 25 to 35 YS and stored at

-70°C, or they were used as fresh YS without freezing.Harvest and purification of C. burnetii The

rickettsiae from the infected YS were purified byisopycnic Renografin gradients as outlined in a pre-vious study (62). Some rickettsiae, however, were pur-ified through gradients containing 0.25 M sucrose,which was also present in buffer components (phos-phate-buffered saline-sucrose, pH 7.35) used for resus-pending the pellets throughout purification.

light microscopy. The Renografin-purified rick-ettsiae (CB9MI) were inactivated with 1% formalde-hyde for 24 h at room temperature. Formaldehyde wasremoved by washing the organisms three times withsterile deionized distilled water at 12,100 x g for 30min. The pellet was resuspended in sterile deionizeddistilled water and applied to the glass slides with aloop to avoid overlap of the bacterial cells in thesample. All samples were air dried, heat fixed, andcooled before staining. The following staining proce-dures were employed. (i) Gram staining was carriedout as described by Gimenez (21), employing mordantsof either aqueous or alcoholic solutions. (ii) The acid-fast staining was carried out by the Kinyoun carbol-fuchsin method (17). (iii) The spore-staining procedurewas a modification of the methods of Dorner (17) andWirtz-Conklin (47). Samples were examined under oilimmersion field with a Zeiss standard 18 light micro-scope.

Electron microscopy. Rickettsiae in YS and frompurified rickettsial suspensions were fixed overnight at40C in a primary fixative containing 2.5% glutaralde-hyde (Polysciences, Warrington, Pa.), 2.0% formalde-hyde prepared from paraformaldehyde (Baker, Phil-lipsburg, N.J.), and 2.5mM CaCl2 in 66mM cacodylatebuffer (pH 6.8). After a brief rinse with buffer, thespecimens were postfixed in 1% osmium tetroxidebuffered with 66 mM cacodylate buffer for 1 h at 40C.The suspensions in microsample tubes (1.5 ml) werecentrifuged (Beckman Microfuge B) after each stageof both fixation and rinsing. The pellets werepreembedded in 2% Difco Noble agar (23), and theblocks were dehydrated through serial dilutions ofmethanol. The cells were stained for 1 h at roomtemperature with 0.5% uranyl acetate in 30% methanolduring dehydration.The nucleoids of C. burnetii were preserved by a

procedure which was described by Ryter and Kellen-berger (52). The Ryter-Kellenberger fixative (30) con-taining 1% osmium tetroxide was also employed as analternative primary fixative. In this instance, the pur-ified sample was fixed by adding 10 volumes of Ryter-Kellenberger fixative (pH 6.1) for 16 h at 40C. Fixationwas followed by three 15-min rinses in Ryter-Kellen-berger fixative Veronal-acetate buffer (pH 6.1). Aftereach stage of fixation and rinsing, the suspension wascentrifuged at 9,750 x g for 30 min at 40C. The pelletwas resuspended and stained with 0.5% uranyl acetatein Ryter-Kellenberger fixative Veronal buffer (pH 6.1)for 2 h at room temperature. After one rinse in buffer,the samples were embedded in 2% Difco Noble agar(23), cut into 1-mm3 blocks, and dehydrated throughserial dilutions of methanol. All blocks were embeddedin Spurr epoxy resin (54). Ultrathin sections were cuton a Reichert OMU2 microtome, collected on un-coated Pelco 300 grids, stained with either lead citrate

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DEVELOPMENT CYCLE OF C. BURNETII 1065

(50) or potassium permanganate (37, 56, 65), and ex-amined in a Hitachi EM HU-11E-1 electron micro-scope operated at 75 kV.

Samples purified with phosphate-buffered saline-sucrose as the diluent (62) were fixed with primaryfixative containing the following components: 3% glu-taraldehyde and 2.5 mM CaCl2 in 66 mM cacodylatebuffer (pH 6.8). Postfixation was carried out with 1%osmium tetroxide buffered with 66 mM cacodylate(pH 6.8). The osmolarity of this buffer and the buffer(66 mM cacodylate, pH 6.8) used for washing afterfixation was adjusted from 137 mOsmol/kg to approx-imately 540 mOsmol/kg with the addition of sucrose(0.37 M). The osmolarity of solutions was determinedby the freezing point depression method with an os-mometer (model 3W, Advanced Instrument, NeedhamHeights, Mass.). Ryter-Kellenberger fixative was usedas an alternative primary fixative (30).

Biochemical assay: dipicolinic acid. The color-imetric assay for dipicolinic acid in bacterial sporeswas carried out by the method of Janssen et al. (28),employing a dipicolinic acid (Sigma Chemical Co., St.Louis, Mo.) standard curve as a positive control.

RESULTSStaining of C. burnetii. The following ob-

servations were made on CB9MI purified byRenografin gradients (62).

(i) Gram-staining. A variable Gram stainreaction was obtained with ethyl-alcohol iodinesolution as a mordant, whereas aqueous iodinegave a gram-negative reaction. Such findingsagree with those of Gimenez (21).

(ii) Acid-fast stain. Approximately 10% ofthe organisms were stained red or were "acidfast," suggesting that C. burnetii cells exhibitsome of the staining properties of tubercle ba-cini.

(iii) Spore stains. With the Wirtz-Conklinmethod, faint-green spherules and rods werebarely distinguishable in a field of red-stainedrods. Clear visualization of the green-stainingbodies, characteristic of spores, was limited dueto the resolution of the light microscope. Withthe Domer method, most cells were stained red,characteristic of spores, whereas bacterial cellsappeared colorless against a dark grey back-ground.Pleomorphism of C. burnetii before and

after Renografin purification: a compari-son of lead-stained thin sections. Morpho-logical features of C. burnetii growing in YS ofan infected embryo is shown in Fig. 1A. At leasttwo morphological cell types, designated asLCVs and SCVs, of C. burnetii were observed.Some cells appeared to be undergoing cellulardeterioration, as evidenced by loose outer cellmembranes and fibrillar nuclear regions (Fig.1A, arrows), whereas other cells were more elec-tron dense (Fig. 1A).

After the long, arduous Renografin purifica-

tion procedure (62), similar morphological cellvariants were observed in the final, highly con-centrated preparation (Fig. 1B). These cells re-sembled the starting YS material; however, celldamage was apparent, as shown by the looseouter membrane and apparent outer membranebleb formation (Fig. 1B, arrows). Morphologicalfeatures of the SCVs appeared to be unaffectedduring the purification procedures (40). Indeed,in an earlier study, we showed that the LCVswere sensitive to decreasing osmotic conditions,whereas the SCVs were not affected by extremesof osmotic pressures and sonic disruption (40).The SCVs were compact and rod-shaped with avery dense central region of condensed nucleoidfilaments (Fig. 1B). This nucleoid region wassurrounded by an electon-dense and granularmaterial, presumably ribonucleoprotein (Fig.1C). The outer membrane of the SCV was barelyresolvable, yet a dense layer beneath the outermembrane, possibly a peptidoglycan, was veryprominent (Fig. 1C). Staining with lead, how-ever, was too intense to allow reasonable visual-ization of the cytoplasmic membrane (Fig. 1A,B, and C).The LCVs, which resembled gram-negative

bacteria, were larger and more pleomorphic thanthe SCVs (Fig. 1). A slightly swollen, and per-haps damaged, LCV is depicted in Fig. 1C, show-ing a loose outer membrane and cytoplasmicmembrane. The nucleoid region, recognized bydense filaments radiating from the central regioninto the cytoplasm, appeared to be more dis-persed in the LCV than in the SCV. Also, theouter membrane of the LCV was more clearlyseparated from the cytoplasmic membrane bythe periplasmic space, whereas the characteristicdense layer of the SCV underlying the outermembrane was not observed. The ribonucleo-protein material was displaced to the peripheryof the cell, leaving a transparent zone betweenthe nucleoid mass and the granular cytoplasm.Comparison ofRenografin-purified LCVs

and SCVs stained with potassium perman-ganate. Morphological features of C. burnetiigrowing in the phagolysosome of an infected YScell is depicted in Fig. 2A. Extreme cellularpleomorphism was observed in this thin section,demonstrating the heterogeneity of cell types.In general, these potassium permanganate-stained cells show similar morphological fea-tures as those described in lead-stained thinsections at low magnification (compare Fig. 1Aand 2A). Importantly, some cells (Fig. 2A) ap-peared to be undergoing cellular deterioration,as evidenced by loose cell membranes and in-creased periplasmic space; however, some LCVswere undergoing cell division with septate for-mation (Fig. 2, arrows).

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FIG. 1. Electron micrographs ofthin sections ofC. burnetii cells observed in situ in YS and after Renografinpurification. C. burnetii cells were fixed withprimary fixative and stained with lead. (A) Demonstration ofthepleomorphic nature of C. burnetii in situ in YS before Renografin purification. Both the LCV (L) and the SCV(S) are clearly depicted. The LCV appears to be undergoing deterioration since, in some cells, the outermembrane is distended, thereby increasing the periplasmic space (arrows). Bar, 0.6 pm. (B) Renografin-purified C. burnetii cells, showing both the LCVs and the SCVs. The LCVs appear to be damaged by thepurification procedure. The outer membrane of the LCVs appears to form blebs (arrows). The morphology ofthe SCVs appears to be unaffected by purification. Bar, 0.6 pm. (C) Resolution of LCV ultrastructure,employing primary fixative and lead staining. The cytoplasmic membrane (CM) and outer membrane (OM)are separated by a periplasmic space (PS). DL, Dense layer. The nucleoplasm, with dispersed nucleoidfilaments and ribonucleoprotein, is clearly shown. Bar, 0.2 pm.

Morphological differences between the cellvariants were more striking (Fig. 2B) at highermagnifications when potassium permanganatestaining was carried out on Renografin-purifiedC. burnetii. Sections of the LCVs and SCVsshowed a well-defined outer membrane and arecognizable cytoplasmic membrane. However,the SCVs are markedly different from the LCVs.Notably, a dense material filled the periplasmicspace between the outer and cytoplasmic mem-branes (Fig. 2B, C, and D), and multilayeredtrilaminar membranes were also observed withinthe cytoplasm of the SCVs (Fig. 2C and D,arrows). These membranes consisted of distinctelectron-lucent layers separated by electron-dense layers. In both transverse (Fig. 2C) andlongitudinal (Fig. 2D) sections, the membranesappeared mostly on one hemisphere of the cell.The outer membrane was 4.5 nm thick, whereasthe internal membranes were 3.5 nm thick. Thecomplex intracellular membranes appeared to

be continuous with the cytoplasmic membrane,and each set of two electron-lucent layers sepa-rated by an electron-dense layer seemed to beinfoldings of a unit membrane. These mem-branes were so closely compacted that the con-tiguous layers, in some cells, were unresolvable.The LCVs, in contrast to the SCVs, did notappear to have internal membranes. Instead, theLCV cell wall consisted simply of a trilaminarcytoplasmic membrane separated from a three-layered outer membrane by a periplasmic space.The peptidoglycan layer was not readily visible.This is in sharp contrast to the electron-densematerial found in the periplasmic space of theSCV.Sporogenic differentiation. Although elec-

tron microscopic studies cannot simulate time-lapse sequence of cellular differentiation, wehave arranged selected photomicrographs of apurported differentiation progression (Fig. 3).The interior of the cell was composed of a very

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FIG. 2. Comparison of thin sections of LCVs and SCVs of C. burnetii in situ in the phagolysosome andafter Renografin purification. The cells were fixed with primary fixative and stained with potassiumpermanganate. (A) Demonstration of the pleomorphic nature of C. burnetii growing in the phagolysosome.Both cell variants are clearly depicted. Some of the LCVs were apparently undergoing deterioration, asevidenced by a loose outer membrane, increased periplasmic space, and outer membrane bleb formation.However, some LCVs are apparently dividing since they are undergoing binary transverse division withseptate formation (arrows). The SCVs appear as extremely dense organiss. Bar, 0.6 gm. (B) Comparison ofLCVs (L) and SCVs (S) ofC. burnetii at higher magnification after-Renografinpurification. Note the distendedappearance of the LCVs and the membrane bleb forma,tion (arrow). The small cell variants appear as densebodies with endogenous membranous instrusions. Bar, 0.2 pm. (C) Higher magnification of transverse sectionofSCVs of C. burnetii after Renografin purification. The cytoplasmic membrane (CM), membranous intrusion(MI), and outer membrane (OM) are clearly definable. DL, Dense layer; R, ribonucleoprotein. Note the densityof the periplasmic space. Bar, 0.2 tum. (D) Longitudinal section of C. burnetii, illustrating the compactness ofthe SCV. The membranous intrusions (MI) are barely recognizable. Note the density of the periplasmic spaceand nucleoplasm. Several layers of membranous intrusions can be observed beneath the cytoplasmic mem-brane. The periplasmic space of the LCV is less dense (compare Fig. I C). Bar, 0.2 pm.

dense nucleoplasm. As the purported differentia-tion progressed, fibrils radiated from the nucleo-plasm, and the dense granules or ribonucleopro-teins formed a tight package surrounding thenucleoplasm (Fig. 3A). Thus, the cell appearedto differentiate into a sporogenic phase with thefollowing features. (i) In some cells, an unusualmorphological feature was observed. Initial ob-servations suggested that an endogenous struc-ture could be shown in one pole of the LCV.Furthermore, cells purified with phosphate-buffered saline as diluent showed fewer of theseultrastructural features than did those cells pu-rified with phosphate-buffered saline-sucrose asdiluent. Therefore, cells were purified in phos-

phate-buffered saline-sucrose as diluent, and po-tassium permanganate-stained thin sectionswere examined under high magnification. In-deed, an endogenous structure was observed de-veloping in one pole of the LCV (Fig. 3A). Thestructure appeared as a "cap" with electron-dense material surrounded by a single trilaminarmembrane (Fig. 3A). The membrane resembledthe cytoplasmic membrane, but the infolding ofthe membrane surrounding the cap did not ap-pear to be continuous with the cytoplasmicmembrane. The space between the cap and thecytoplasmic membrane seemed to share the areawith the periplasmic space (Fig. 3A and B). (ii)The LCV appeared to undergo a stage in vege-

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tative differentiation which corresponded to en-dospore formation during a process of unequalcell division (Fig. 3B). The cap structure ap-peared to be initiated in an intermediate stageof SCV to LCV differentiation, thus proceedingto an endogenous structure characteristic of spo-rogenic differentiation. (iii) As the progressioncontinued, an endospore was clearly depicted inone pole of the LCV (Fig. 30). The coat of thedense body or endospore was approximately 26.4+ 2.5 nm in thickness and consisted of threeelectron-lucent layers separated by two electron-dense areas (Fig. 30). (iv) Separation of thedense body from the main cytoplasmic compo-nent of the cell is depicted in Fig. 3D. The LCVcontaining the endospore appeared to be dete-riorating, suggesting that the endospore may beliberated upon lysis of the LCV.The purification procedure probably contrib-

uted to the distended appearance of the LCVscontaining endospores. Since we observed thesemarked increases in the periplasmic space ofsome cells (compare Fig. 2A and 3), it is conceiv-able that some cellular damage was promotedby the purification procedure. However, theLCVs showed extreme pleomorphism in situduring intraphagolysosomal growth (Fig. 2A). Itis worth noting that the LCVs in situ appearedto deteriorate, shedding cytoplasmic compo-nents as well as the cell envelope material.Nuclear region ofthe endospore. Since the

endospore could be visualized in those cellswhich had been fixed with potassium perman-ganate, it was of interest to demonstrate thepurported nuclear regions. We therefore usedthe Ryter-Kellenberger procedure (52) in an at-tempt to demonstrate subcellular nucleic acidbounded by membranes (Fig. 4A). This methodshowed at least one electron-dense body sur-rounded by an electron-lucent zone within sev-eral LCVs (Fig. 4A and B, arrows). Since theRyter-Kellenberger procedure had been shownto stabilize the nuclear material ofbacterial cells,the extreme density of the subcellular structuresmay reflect an aggregation of nuclear material

enclosed in membranes within the boundary ofthe LCVs. The subcellular orientation of theendospore in purified C. burnetii cells was dem-onstrated by the Ryter-Kellenberger (Fig. 4B)and potassium permanganate (Fig. 40) methods.Similar subcellular structures were observedwhen the potassium permanganate procedurewas carried out on C. burnetii, phase I or II, insitu in chicken embryo tissue culture cells (Fig.4D).Mode of propagation of C. burnetii. There

are at least two modes of propagation of C.burnetii. Binary transverse fission was observedto occur in both the SCVs and LCVs (Fig. 5).Transverse septate formation appeared to occurin both cell variants during cell division (Fig. 5Aand B, arrows); however, some LCVs appearedto undergo cell division without septate forma-tion (Fig. 50). Some LCVs were observed toundergo concurrent binary transverse fissionwith unequal cell division characterized by en-dospore formation (Fig. 30).Biochemical assay for dipicolinic acid.

The assay for dipicolinic acid, employing 50 mg(dry weight) of Renografin-purified rickettsialsuspension of CBOI per ml, was negative (28).

DISCUSSIONThe observations outlined in this study were

made on C. burnetii cells before and after Ren-ografin purification. Although extreme pleomor-phism was noted in the purified cells, similarmorphological properties of C. burnetii wereobserved in the phagolysosome of infected eu-caryotic cells. Our results can be explained onthe basis of differences ofmorphology oftwo cellvariants of C. burnetii. The LCVs appeared sim-ilar to the gram-negative bacteria in possessingouter and cytoplasmic membranes separated bya periplasmic space. The SCVs, on the otherhand, had an extremely electron-dense area be-tween the cytoplasmic and outer membranesand a well-defined membranous system, possiblycontinuous with the cytoplasmic membrane. Atthe ultrastructural level, the combination of

FIG. 3. Purported differentiation progression of endospore formation by C. burnetii. Selected photomicro-graphs from Renografin-purified C. burnetii cells were arranged in a proposed differentiation progression.Cells were fixed with primary fixative and stained with potassium permanganate. (A) Initial stages ofendospore formation within thepolaC end ofthe large cell variant. Note theperiplasmic orientation ofthe capformation (arrow). PS, Periplasmic space. Bar, 0.2 pm. (B) Intermediate stage ofendospore formation showingthe polar and periplasmic orientation of endospore development during a process of unequal cell division.The periplasmic space is greatly distended, probably as a result ofpurification. Bar, 0.2 pm. (C) Completeformation of the endospore (E) in an LCV concurrently undergoing unequal cell division. Note the nuclearregions of the dividing cell and the separation ofthe spore from cytoplasmic contents by the membranes oftheendospore. Bar, 0.2 pm. (D) Apparent degeneration ofan LCV containing an endospore (E). Bar, 0.2 ,um. Theendospore may be released from the LCV upon complete lysis of the cell.

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E

FIG. 4. Comparison of electron micrographs of thin sections of C. burnetii cells fixed and stained by theRyter-Kellenberger andpotassium permanganate methods. (A) Nucleoid region ofthe endospore (E) is clearlyvisible in electron micrographs of thin sections of C. burnetii fixed with Ryter-Kellenberger fixative andstained with lead. Note two LCVs with endospores (E) (arrows). Bar, 0.4 tum. (B, C, and D) Highermagnification illustrating the polar location and the density of the endospore (E), reflecting the limitingmembranes of the endospore and aggregation of nucleic acid. Bar, 0.2 pn. (B) Ryter-Kellenberger procedure,(C) potassium permanganate procedure, and (D) potassium permanganate procedure carried out in situ inchicken embryo tissue culture cells infected with C. burnetii, phase I and II. In (D), the cell was in phase IIand the surface was coated with antibody (manuscript in preparation).

macromolecules and peptidoglycan may contrib-ute to an electron-dense area in the cell walls ofthe SCVs. The peptidoglycan was not as obviousin the LCVs.

Such vast differences in morphology betweenLCVs and SCVs provide some possible clues insolving the puzzling phenomena ofboth the vari-able Gram stain reaction of C. burnetii and the

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FIG. 5. Comparison of ultrasections of C. burnetii showing cell division with septate formation of the LCVsand SCVs. (A) SCV fixed with primary fixative and stained with potassium permanganate. (B) LCV fixedwith primary fixative and stained with potassium permanganate. (C) LCVfixed with the Ryter-Kellenbergertechnique and stained with lead. The surfaces of the organisms in both (A) and (C) are coated with antibody(in preparation). Although the SCV undergoes transverse binary fission, the frequency was low in thesestudies. Bar, 0.2 pm.

relationship between the two cell variants. C.burnetii is generally regarded as gram negative(29), but under certain conditions of staining, itcan be shown to be gram positive, as demon-strated in this and a previous study (21). Bio-chemically, muramic acid (35) and diaminopi-melic acid (41) have been detected in the cells ofC. burnetii, although the concentration of dia-minopimelic acid was lower than that reportedfor gram-negative bacteria (41). Lipopolysaccha-ride has been isolated from C. burnetii andshares properties with that of gram-negativebacteria (5, 6). The structure of the cell wall, thecomposition and possession of the muramic anddiaminopimelic acid components, and the pres-

ence of lipopolysaccharide suggest that the cellwalls of C. burnetii resemble those of gram-negative bacteria. Different views, however,have been expressed by other investigators. Ner-mut et al. (43) observed that the peptidoglycanlayer could only be removed from the outer layerby prolonged treatment with hot trichloroaceticacid. Burton et al. (8) reported a thin, moder-ately electron-dense intermediate layer associ-ated with the inner surface of the outer mem-brane. This intermediate layer was unaffectedby lysozyme and EDTA treatment, which indi-cated that this structure might not be compa-rable to that of the peptidoglycan of gram-neg-ative bacteria. We showed that the SCVs possess

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a complex internal cytoplasmic membranoussystem that appears to consist of several trilam-inar membranes. These membranes may explainthe findings of Burton et al. (8), who describedthree instead of two peripheral membranes insome C. burnetii. They could not determinewhether this third or intermediate membranewas equivalent to the cytoplasmic or the outermembrane. In our studies, cells were observedwith three peripheral membranes that may rep-resent the intermediate stage between the SCVand LCV during a process consistent with vege-tative differentiation (20). Postassium perman-ganate staining in the SCVs revealed a verydense central nucleoid region, showing a fibril-lar network resembling ribonucleoprotein fila-ments.

In contrast to the SCVs, complex membra-nous systems were not observed in the LCVs.Structurally, the LCVs was bounded by both theouter and cytoplasmic membranes, which wereseparated by a periplasmic space. Peptidoglycanwas not clearly observed; however, preliminarychemical analysis of the cell wall shows that theLCVs possess quantitatively less peptidoglycanthan do the SCVs (K. Amano and J. C. Williams,unpublished data). As reported by others (9, 31,42, 61), the central nucleoid filaments in mostLCVs were more dispersed, and the ribonucleo-protein particles were less concentrated and sit-uated more peripherally than in the SCVs. Moreimportant, some LCVs possessed an endogenousstructure that was more clearly observed afterstaining with potassium permanganate. Thus, C.burnetii appears to have an additional mode ofmultiplication in conjunction with transverse bi-nary fission. This additional mode results fromunequal cell division and is unique among anyobligately intracellular parasite, but it is similarto the bacterial endospore differentiation ofgram-positive bacteria (20). The electron-densebody, about 130 to 170 nm in diameter, wassurrounded by a well-defined coat of at leastfour layers. The endospore appeared to developendogenously in the periplasmic space of onepole of LCVs during unequal cell division (Fig.3D). The formation of endospores was also ob-served in both phase I and phase II cells withinthe phagolysosome of infected tissue culturecells (Fig. 4D).Endospores (14) are formed under various ex-

perimental conditions by members ofmany gen-era of bacteria (20), such as Bacillus and Clos-tridium, which are widely distributed, especiallyin soil. An important aspect of the bacterialendospore is the degree of both heat resistanceand survival in nature. C. burnetii is also uniquein that it is more resistant to physical and chem-

ical agents than any other pathogenic rickettsia(45). The heat resistance of C. burnetii is consid-erable. In milk, for instance, C. burnetii is resist-ant for 30 min at 630C and even for 15 s at 740C(4). The resistance to cold is even more remark-able, in that C. burnetii held at -20°C remainedviable for almost 2 years (4). C. burnetii hasbeen known to survive on wool, clay, and sand(27, 60), and its ability to persist outside its hosthas also been shown by isolation from air anddust (16).

Recently, a study was made to test the effectof physical stress on C. burnetii (40). Suspen-sions of organisms were subjected to osmoticshock in water at 450C followed by sonication at40C, incubation at elevated temperatures at450C for 3 h, and finally, centrifugation througha 40 to 70% sucrose density gradient at 40C.Examination of the cells by transmission elec-tron microscopy revealed that the final fractioncontained SCVs which were found to be infec-tious in chicken embryonated eggs, whereas theLCVs were virtually eliminated in the final prep-aration (40). The final product, however, wasshown to retain metabolic activity although ata slower rate than that of the starting material.These data suggest that the SCV of C. burnetiiretains its viability during exposure to extremeenvironmental conditions. Thus, the SCV is aheat-resistant relatively dormant structurewhich has the ability to survive in an adverseenvironment(s).

Bacterial endospores are also known to havestructural characteristics that distinguish themfrom the corresponding growing or vegetativeorganisms. The multilayered membranes andthe dense layer in the cell wall discriminates theSCVs from the vegetative counterparts, theLCVs. Bacterial endospore differentiation pro-ceeds through well-defined stages which aremorphologically identifiable and well docu-mented for Bacillus sp. and Clostridium sp. (3,20, 66, 67). However, the sporulation process forC. burnetii requires further study before we candescribe each phase of sporogenic differentia-tion.

Dipicolinic acid, a common chemical constit-uent of most spores (3), was not detected in C.burnetii cells. However, some cells of C. burnetiiwere stained acid fast by the Kinyoun carbol-fuchsin method, which detects acid-fast bacilli.Apparent differences in morphology of both theSCVs and LCVs stress the need to establish theidentity of chemical components in the cell wallsofeach cell variant and to correlate such findingsto the Gram variability and the acid-fast prop-erties of C. burnetii.Based on the morphology of C. burnetii in

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unsynchronized cultures, we hypothesize a spec-ulative developmental cycle which consists ofvegetative and sporogenic differentiations (Fig.6). This putative developmental cycle of C. bur-netii proposes the morphogenesis of spore for-mation which should be advantageous for themicrobe since it would offer considerable sur-vival value in nature. Although both physiolog-ical and biochemical factors involved in the for-mation and germination of C. burnetii endo-

'9

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spores have not been determined, previousknowledge of the proliferation and metabolismof C. burnetii provides some insight into thesephenomena. Differentation of the spore to thevegetative state may require the participation ofthe host eucaryotic cell since C. burnetii isknown to reside in the phagolysosomal com-partment. This compartment is a hostile envi-ronment in which the microbe is exposed to lowpH (44), hydrolytic enzymes (48), and other

3

ative:nti ation

4

FIG. 6. Schematic ofthe putative developmental cycle of C. burnetii within thephagolysosome ofeucaryoticcells. (1) The developmental cycle may be initiated coincident with engulfment of the spore or SCV by aphagocytic cell. Upon entry of the SCV into the phagolysosome, the acid pH of the phagolysosome mayactivate generalized metabolism of C. burnetii (24, 25, 40). (2) The SCV may undergo multiplication bytransverse binary fission. (3 and 4) Alternatively, the SCV may differentiate to the vegetative cell variant.Changes initiated bypH, enzymatic systems, and/or nutritional status within the phagolysosome may be thetriggering mechanisms that induce vegetative differentiation. At this stage, the multilayered membranesshould become less visible, and the dense nucleoid may begin to disperse. (5) Further differentiation of theLCVmay proceed to a transverse binary division stage. (6) Alternatively, the division stage may coincide withsporogenic differentiation, resulting in unequal cell division. Changes initiated during the progressiveinfection of the eucaryotic cell may signal the LCV to undergo sporogenesis. Indeed, the eucaryotic cellbecomes filled with the phagolysosome containing large numbers of C. burnetii in different stages ofdevelopment (see Fig. 2A). (7, 8 and 9). Putative sequences during the sporogenic differentiation must includethe polar development of the spore ranging from 130 to 170 nm in diameter. (10) Release of the spore from theLCV may occur upon lysis of the LCV. Release of the spore from the LCV may lead to the maturation of theSCV.

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microbicidal mechanisms (22). The signals re-quired to induce differentiation must thereforeinitiate in the phagolysosome of the host cell.The function of the SCV or spore, due to thethick periplasmic wall, is perhaps to survive inthe phagolysosome and in an extracellular en-vironment during aerosol dissemination.

Further, proliferation and multiplication of C.burnetii in the phagolysosome may involve thepH activation of protein and DNA and RNAbiosynthesis (24, 25). Indeed, Hackstadt andWilliams (24) have shown that phagosome-ly-sosome fusion is required to generate conditionsfavorable to C. burnetii multiplication. Growthof C. burnetii in the phagolysosome of the hostcell may inevitably lead to a depletion of essen-tial nutrients or an increase in the pH within thevacuoles. Thus, an alteration in the nutritionalstatus and pH of the phagolysosome may bringabout an induction ofendospore formation. Boththe primary induction of synthesis for sporoge-nesis and initiation of cellular division by binaryfission appear to be independently triggeredsince dividing cells can undergo sporogenesis(Fig. 3). However, binary transverse fission ap-pears to be a common feature for both cellvariants. In Escherichia coli, a large cell thatinitiates DNA replication will proceed faster todivision than cells that initiate at a small size(17). Since the two cell variants of C. burnetiishow obvious size differences, the LCV is morelikely to undergo binary fission than the SCV.

Interesting parallels in ultrastructure and de-velopmental cycle may be drawn between thegenera Chlamydia and Coxiella. However, thefollowing comparison clearly delineates themarked differences which exist between thesegenera. (i) These intracellular rickettsiae occupydifferent compartments within the eucaryoticcell; Chlamydia organisms carry out their de-velopmental cycle in the phagosome (18),whereas C. burnetii growth occurs only in thephagolysosome (24). Indeed, the metabolism ofexogenous substrates by C. burnetii is activatedat acid pH, whereas Chlamydia spp. metabolizesubstrate at neutral pH (24, 25). (ii) Morpholog-ical heterogeneity of both genera has been dem-onstrated in situ in eucaryotic cells and in puri-fied preparations (1, 7, 8, 10-13, 18, 26, 31-36, 40,42, 49, 51). Chlamydia spp. and C. burnetii showa range of particle sizes with varying degrees ofosmotic stability and infectivity. Only the ele-mentary body of Chlamydia organisms is infec-tious (57), whereas for C. burnetii, the LCV,SCV, and filter-passing particles are infectious(33, 40, 61). (iii) Ultrastructural evidence sug-gests that these rickettsiae are quite similar;however, these microorganisms are, in fact, quite

different. Both genera exhibit extreme pleomor-phism during growth in their respective intra-cellular compartments (10-13, 33-36, 51). Moreimportant, Chiamydia organisms lack a well-defined peptidoglycan (10, 19), whereas Coxiellaorganisms possess a clearly distinguishable pep-tidoglycan, as evidenced by chemical analysis oftypical bacterial cell walls (29). Indeed, prelimi-nary fractionation of cell walls and chemicalanalysis suggest to us that the SCVs possess amore complete peptidoglycan than do the LCVs(Amano and Williams, unpublished data). De-tailed ultrastructural studies indicate that Chla-mydia spp. are propagated within phagocyticvacuoles by means of a biphasic developmentalcycle which consists of the transition of infectingelementary bodies to reticulate bodies dividingby binary transverse fission, without apparentseptation, followed by nuclear condensation toform the infectious elementary body (10-13, 18,49). In comparison, some investigators have sug-gested that the genus Coxiella has a complexdevelopmental cycle (33-36, 61) in which twocell types represent separate stages similar tothose of Chlamydia spp. (51). We have illus-trated a putative developmental cycle of C.burnetii, showing a progression through bothvegetative and sporogenic differentiations char-acterized by binary transverse division, with sep-tation, and unequal cell division during endo-spore formation. Thus, there was no evidencefor "condensation" of the LCV or reorganizationof the nucleoid region with the cell that wouldrepresent the differentiation of a reticulate bodyto an elementary body of Chlamydia sp. Litwin(38) and Litwin et al. (39) have discussed thepossible occurrence of endospore formation inChlamydia spp. and contrasted this aberrantdevelopmental cycle to the formation of bacte-rial spores. An interesting parallel might bedrawn if the question of the developmental cycleof Chlamydia sp. were studied again, employingthe techniques used in our study, especially po-tassium permanganate as a membrane stain.The present data, however, indicate marked dif-ferences in the ultrastructures and developmen-tal cycle of C. burnetii and Chlamydia spp.The discovery of sporogenic differentiation by

C. burnetii should facilitate future studies onthe pathogenicity of this highly infectious andresistant bacterium. Physiological and biochem-ical factors involved in the fornation and ger-mination of endospores are currently being eval-uated.

ACKNOWLEDGMENTSThe technical assistance of E. Davis, the expert secretarial

assistance of S. Smaus, and the assistance provided by R.

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Evans and C. Taylor for photography and illustrations aregratefully acknowledged.

LITERATURE CITED1. Anacker, R. L., K. Fukushi, E. G. Pickens, and D. B.

Lackman. 1964. Electron microscopic observations ofthe development of Coxiella burnetii in the chick yolksac. J. Bacteriol. 88:1130-1138.

2. Ariel, B. M., T. N. Khavkin, and N. I. Amosenkova.1973. Interaction between Coxiella burnetii and thecells in experimental Q-Rickettsiosis. Pathol. Microbiol.39:412423.

3. Aronson, A. I., and P. Fitz-James. 1976. Structure andmorphogenesis of the bacterial spore coat. Bacteriol.Rev. 40:360-402.

4. Babubieri, B. 1959. Q fever: a zoonosis. Adv. Vet. Sci. 5:81-182.

5. Baca, 0. G., and D. Paretsky. 1974. Some physiologicaland biochemical effects of a Coxiella burnetii lipopoly-saccharide preparation on guinea pigs. Infect. Immun.9:939-945.

6. Baca, 0. G., and D. Paretsky. 1974. Partial chemicalcharacterization of a toxic lipopolysaccharide from Cox-iella burnetii. Infect. Immun. 9:959-961.

7. Burton, P. R., N. Kordova, and D. Paretsky. 1971.Electron microscopic studies of the rickettsia Coxiellaburnetii: entry, lysosomal response, and fate of rickett-sial DNA in L-cells. Can. J. Microbiol. 17:143-150.

8. Burton, P. R., J. Stueckemann, and D. Paretsky.1975. Electron microscopy studies of the limiting layersof the rickettsia Coxiella burnetii. J. Bacteriol. 122:316-324.

9. Canonico, P. G., M. J. Van Zwieten, and W. A. Christ-mas. 1972. Purification of large quantities of Coxiellaburnetii rickettsia by density gradient zonal centrifu-gation. Appl. Microbiol. 23:1015-1022.

10. Costerton, J. W., L. Poffenroth, J. C. Wilt, and N.Kordova. 1975. Ultrastructural studies of Chlamydiapsittaci 6BC in situ in yolk sac explants and L cells: acomparison with gram-negative bacteria. Can. J. Micro-biol. 21:1433-1447.

11. Costerton, J. W., L. Poffenroth, J. C. Wilt, and N.Kordova. 1975. The effect of purification on the ultra-structure and infectivity of egg-attenuated Chlamydiapsittaci (6BC). Can J. Microbiol. 21:1449-1463.

12. Costerton, J. W., L. Poffenroth, J. C. Wilt, and N.Kordova. 1976. Alterations in the ultrastructure ofChlamydia psittaci 6BC harvested from the allantoicfluid of chick embryos. Can. J. Microbiol. 22:9-15.

13. Costerton, J. W., L. Poffenroth, J. C. Wilt, and N.Kordova. 1976. Ultrastructural studies of the nucleoidsof the pleomorphic forms of Chlamydia psittaci 6BC:a comparison with bacteria. Can. J. Microbiol. 22:16-28.

14. Cross, T., and R. W. Attwell. 1975. Actinomycetespores, p. 3-14. In P. Gerhardt, R. N. Costilow, and H.L. Sadoff (ed.), Spores VI. American Society for Micro-biology, Washington, D.C.

15. Davis, G. E., and H. R. Cox. 1938. A filter-passinginfectious agent isolated from ticks. Public Health Rep.53:2259-2267.

16. Delay, P. D., E. H. Lennette, and K. B. DeOme. 1950.Q fever in California. II. Recovery of Coxiella burnetiifrom naturally-infected airborne dust. J. Immunol. 65:211-220.

17. Finegold, S. M., W. J. Martin, and E. G. Scott. 1978.Staining formulas and procedures, p. 472-480. In W. R.Bailey and E. G. Scott (ed.), Diagnostic microbiology,5th ed. C. V. Mosby Co., St. Louis.

18. Friis, R. R. 1972. Interaction of L cells and Chiamydiapsittaci: entry of the parasite and host responses to itsdevelopment. J. Bacteriol. 110:706-721.

19. 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.

20. Ghosh, B. K., and A. Ghosh. 1977. Introduction to theultrastructure of bacteria, p. 43-117. In A. I. Laskin andH. A. Lechevalier (ed.), CRC handbook of microbiology,vol. 1, Bacteria. CRC Press, Boca Raton, Fla.

21. Gimenez, D. F. 1965. Gram staining of Coxiella burnetii.J. Bacteriol. 90:834-835.

22. Goren, M. B. 1977. Phagocyte lysosomes: interactionswith the infectious agents, phagosomes and experimen-tal perturbations in function. Annu. Rev. Micriobiol.31:507-533.

23. Gowans, E. J. 1973. An improved method of agar pelletedcells for electron microscopy. Med. Lab. Technol. 30:113-115.

24. Hackstadt, T., and J. C. Williams. 1981. Biochemicalstratagem for obligate parasitism of eukaryotic cells byCoxiella burnetii. Proc. Natl. Acad. Sci. U.S.A. 78:3240-3244.

25. Hackstadt, T., and J. C. Williams. 1981. Incorporationof macromolecular precursors by Coxiella burnetii inan axenic medium. In W. Burgdorfer and R. L. Anacker(ed.), Rickettsiae and rickettsial diseases, AcademicPress, Inc., New York.

26. Handley, J., D. Paretsky, and J. Stueckemann. 1967.Electron microscopic observations of Coxiella burnetiiin the guinea pig. J. Bacteriol. 94:263-267.

27. Ignatovich, V. F. 1959. A contribution to the question ofthe retention of Rickettsia burnetii on objects of theexternal environment. J. Microbiol. Epidemiol. Immu-nol. 30:156-157.

28. Janssen, F. W., A. J. Lund, and L. F. Anderson. 1958.Colorimetric assay for dipicolinic acid in bacterialspores. Science 127:26-27.

29. Jerrels, J. R., D. J. Hinrichs, and L. P. Mallavia. 1974.Cell envelope analysis of Coxiella burnetii phase I andphase II. Can. J. Microbiol. 20:1465-1470.

30. Kellenberger, E., A. Ryter, and J. Sechaud. 1958.Electron microscope study of DNA-containing plasma.II. Vegetative and mature phage DNA as comparedwith normal bacterial nucleoids in different physiologi-cal states. J. Biophys. Biochem. Cytol. 4:671-678.

31. Kishimoto, R. A., B. J. Veltri, P. G. Canonico, F. G.Shirey, and J. S. Walker. 1976. Electron microscopicstudy on the interaction between normal guinea pigperitoneal macrophages and Coxiella burnetii. Infect.Immun. 14:1087-1096.

32. Kishimoto, R. A., B. J. Veltri, F. G. Shirey, P. G.Canonico, and J. S. Walker. 1977. Fate of Coxiellaburnetii in macrophages from immune guinea pigs.Infect. Immunol. 15:601-607.

33. Kordova, N. 1959. Filterable particles of Coxiella bur-netii. Acta Virol. 3:25-36.

34. Kordova, N. 1960. Study of antigenicity and immunogen-icity of filterable particles of Coxiella burnetii. ActaVirol. 4:56-62.

35. Kordova, N. 1978. Chlamydiae, rickettsiae and their cellwall defective variants. Can. J. Microbiol. 24:339-352.

36. Kordova, N., and E. Kovacova. 1968. Appearance ofantigens in tissue culture cells inoculated with filterableparticles of Coxiella burnetii as revealed by fluorescentantibodies. Acta Virol. 12:460-463.

37. Lawn, A. M. 1960. The use of potassium permanganateas an electron-dense stain for sections of tissues embed-ded in epoxy resin. J. Biophys. Biochem. Cytol. 7:197-198.

38. Litwin, J. 1959. The growth cycle of the psittacosis groupsof microorganisms. J. Infect. Dis. 105:129-160.

39. Litwin, J., J. E. Officer, A. Brown, and J. W.Moulder. 1959. A comparative study of the growth

VOL. 147, 1981


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cycles of different members of the psittacosis group indifferent hosts cells. J. Infect. Dis. 109:251-279.

40. McCaul, T. F., T. Hackstadt, and J. C. Williams. 1981.Ultrastructural and biological aspects of Coxiella bur-netii under physical disruptions. In W. Burgdorfer andR. L. Anacker (ed.), Rickettsiae and rickettsial diseases,Academic Press, Inc., New York.

41. Myers, W. F., R. A. Ormsbee, J. V. Osterman, and C.L. Wisseman, Jr. 1967. The presence of diaminopi-melic acid in the rickettsia. Proc. Soc. Exp. Biol. Med.125:459-462.

42. Nermut, M. V., S. Schramek, and R. Brezina. 1968.Electron microscopy of Coxiella burnetii phase I andII. Acta Virol. 12:446-452.

43. Nermut, M. V., S. Schramek, and R. Brezina. 1972.Further investigations on the fine structure of Coxiellaburnetii. Zentralbl. Bakteriol. Hyg. I. Abt. Orig. A 219:211-226.

44. Ohkuma, S., and B. Poole. 1978. Fluorescence probemeasurement of the intralysosomal pH in living cellsand the perturbation of pH by various agents. Proc.Natl. Acad. Sci. U.S.A. 75:3327-3331.

45. Ormsbee, R. A. 1965. Q fever rickettsia, p. 1144-1160. InF. L. Horsfall and I. Tamm (ed.), Viral and rickettsialdiseases of man. J. B. Lippincott, Philadelphia.

46. Ormsbee, R. A. 1969. Rickettsiae (as organisms). Annu.Rev. Microbiol. 23:275-292.

47. Paik, G., and M. T. Suggs. 1974. Reagents, stains andmiscellaneous test procedures, p. 930-950. In E. H.Lennette and E. H. Spaulding (ed.), Manual of clinicalmicrobiology, 2nd ed. American Society for Microbiol-ogy, Washington, D.C.

48. Pitt, D. 1975. Integrated themes in biology, lysosomes andcell function. Longman Inc., New York.

49. Poffenroth, L., J. W. Costerton, N. Kordova, and J.C. Wilt. 1973. Ultrastructural studies of Chlamydiapsittaci 6BC variant strains. I. Ultrastructure of thesurface layers of egg-passaged 6BC strain. Can. J. Mi-crobiol. 19:887-894.

50. Reynolds, E. S. 1963. The use of lead citrate at high pHas an electron opaque stain in electron microscopy. J.Cell. Biol. 17:208-212.

51. Rosenberg, M., and N. Kordova. 1960. Study of intra-cellular forms of Coxiella burnetii in the electron mi-croscope. Acta Virol. 4:52-55.

52. Ryter, A., and E. Kellenberger. 1958. Etude au micro-scope electronique de plasma contenant de l'acide de-soxyribonucleique. I. Les nucleoiodes des bacteries encroiasance active. Z. Naturforsch. 13B:597-605.

53. Schramek, S. 1968. Isolation and characterization of de-

oxyribonucleic acid from Coxiella burnetii. Acta Virol.12:18-22.

54. Spurr, A. R. 1969. A low-viscosity epoxy resin embeddingmedium for electron microscopy. J. Ultrastruct. Res.26:31-43.

55. Stoker, M. G. P., and P. Fiset. 1956. Phase variation ofthe Nine Mile and other strains of Rickettsia burnetii.Can. J. Microbiol. 2:310-321.

56. Sutton, J. R. 1968. Potassium permanganate staining ofultra thin sections for electron microscopy. J. Ultra-struct. Res. 21:424-429.

57. Tamura, A., A. Matsumoto, and N. Higashi. 1967.Purification and chemical composition of reticulatebodies of the meningopneumonitis organisms. J. Bac-teriol. 93:2002-2008.

58. Wachter, R. F., G. P. Briggs, J. D. Gangemi, and C.E. Pedersen, Jr. 1975. Changes in buoyant densityrelationships oftwo cell types of Coxiella burnetii phaseI. Infect. Immun. 12:433-436.

59. Weiss, E. 1973. Growth and physiology of rickettsiae.Bacteriol. Rev. 87:259-283.

60. Welsh, H. H., E. H. Lennette, F. R. Abinanti, J. F.Winn, and W. Kaplan. 1959. Q fever studies. XXI.The recovery of Coxiella burnetii from the soil andsurface water of premises harboring infected sheep. Am.J. Hyg. 70:14-20.

61. Wiebe, M. E., P. R. Burton, and D. M. Shankel. 1972.Isolation and characterization of two cell types of Cox-iella burnetii phase I. J. Bacteriol. 110:368-377.

62. Williams, J. C., M. Peacock, and T. F. McCaul. 1981.Immunological and biological characterization of Cox-iella burmetii, phases I and II, separated from hostcomponents. Infect. Immun. 32:840-851.

63. Wisseman, C. L., Jr. 1968. Some biological properties ofrickettsiae pathogenic for man. Zentralbl. Bakteriol.Parasitenk. Infektionskr. Hyg. Abt. Orig. 206:299-313.

64. Wyatt, G. R., and S. S. Cohen. 1952. Nucleic acids ofrickettsiae. Nature (London) 170:846447.

65. Yasutake, S., M. Murakami, and N. Yoshida. 1961.Double staining of tissue sections for electron micros-copy with heavy metals KMnO4 and PG (CH3COO)2. J.Electron Microsc. 10:233-237.

66. Young, L. E., and P. C. Fitz-James. 1959. Chemical andmorphological studies of bacterial spore formation. I.The formation of spores in Bacillus cereus. J. Biophys.Biochem. Cytol. 6:467-482.

67. Young, I. E., and P. C. Fitz-James. 1959. Chemical andmorphological studies of bacterial spore formation. II.Spore and parasporal protein formation in Bacilluscereus var. Alesti. J. Biophys. Biochem. Cytol. 6:483-498.

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