jan. p. vol. ©1968 american prinrted ultrastructure...

JOURNAL OF VIROLOGY, Jan. 1968, p. 56-68Copyright © 1968 American Society for Microbiology

Vol. 2, No. 1Prinrted in U.S.A.

Ultrastructure of Bacterial Cells Infected withBacteriophage PM2, a Lipid-containing

Bacterial VirusEUGENE COTA-ROBLES, ROMILIO TORRES ESPEJO,1 AND PATRICIA WILLIAMS HAYWOOD

Departmenit of Life Sciences, Unziversity of California, Riverside, California 92502

Received for publication 12 September 1967

The cytological pattern of infection of a host pseudomonad with PM2, a lipid-containing bacterial virus, was investigated by electron microscopy. Normal andinfected cells frequently contain a myelin figure, which is found in the nucleoidregion or at the periphery of the cell. The most striking finding in this investigationwas that completed virions are found in the cell adjacent to or in association withthe cytoplasmic membrane. This localization is precise; virions are not foundelsewhere in infected cells. The completed virions occasionally appear to be at-tached to the cytoplasmic membrane. The virus contains a darkly staining core

surrounded by a tripartite envelope of a thickness of approximately 70 A, which isidentical to the thickness of the cytoplasmic membrane. Lysing cells appear toundergo extensive damage of the cytoplasmic membrane prior to rupture of the Llayer of the cell wall.

Two marine bacteriophages active againstPseudomonas species were shown to be markedlyinactivated by organic solvents (9). The sensitivityof bacterial viruses to organic solvents is not aproperty heretofore recognized as significantamong bacteriophages; in fact, resistance tochloroform is commonly utilized to facilitate thestudy of the adsorption of bacteriophages totheir host (10). It must be remembered thatobligately parasitic procaryotic cells such asBdellovibrio (11), which form plaques on lawns ofappropriate host bacteria, are inactivated byorganic solvents. Thus, the inhibition of plaque-forming ability by chloroform must not neces-sarily be construed as the inactivation of a bac-terial virus by an organic solvent. However, it istrue that a number of animal viruses are inacti-vated by organic solvents (2), and with certain ofthese viruses it has been clearly shown that someof those viruses which contain phospholipids arereadily disrupted by lipid solvents (3).

Espejo and Canelo (submitted for publication)recently described the chemical and physicalproperties of a unique deoxyribonucleic acid-con-taining bacteriophage, PM2, which infects amarine pseudomonad. These workers showed that

1 Participant in University of Chile-University ofCalifornia Cooperative Program. Permanent ad-dress: Faculty of Sciences, University of Chile, San-tiago, Chile.

PM2, which contains up to 10% of its dry weightas lipid, is inactivated by ethyl ether and is dis-rupted by dilute solutions of the detergentSarkosyl. Electron microscopy of PM2 revealsthat this virus possesses a polyhedral structurewith a diameter of 600 A. PM2 stained withuranyl acetate frequently reveals an apparentlydouble-layered envelope surrounding an innercore.The presence of appreciable levels of lipid in

PM2, plus the "apparent" envelope seen byelectron microscopy, suggested that PM2 mightbe a bacteriophage counterpart of animal virusessuch as influenza virus or Newcastle disease virus.The structure of such viruses has been studied ex-tensively in thin sections by electron microscopy.It was suggested by Hoyle (4) that such virusesderive their outer coat from the cytoplasmicmembrane of the host cell. The electron micro-scopic studies of Morgan et al. (7) offered con-vincing support to Hoyle's suggestion.

This report describes the ultrastructure of hostpseudomonad cells during the replication of PM2,the structure of the virus within infected cells, andthe pattern of cell lysis leading to viral release. Wehave observed a pattern of viral distributionwithin infected cells which is quite different fromthat recently described by Valentine and Chap-man (12) for the bacteriophage NCMB 385 whichinfects another marine bacterium, Cytophagamarinoflava.

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PM2, A LIPID-CONTAINING PHAGE

MATERIALS AND METHODS

Cultivationi of bacteria anzd bacteriophage. Thehost bacterium, which has been tentatively identifiedas a member of the P. stuzerii subgroup, was routinelycultivated with forced aeration at 29 C in a mediumcontaining 8 g of dehydrated nutrient broth per literof AMS solution. AMS solution contains 0.7 g ofKCl, 1.5 g of CaCl2 2H20, 12.0 g of MgSO4 12H20,and 26.0 g of NaCl per liter of distilled water. Thebacterial cells were cultured to a population densityof 3 X 108 cells per ml, at which point they were in-fected with lysates of PM2 at a multiplicity of infec-tion of 5 to 10. In such infected cultures, lysis beginsat about 60 min after infection and is complete by 90min after infection.

Electron microscopy. Samples of infected cells wereremoved at 30, 45, 60, 70, 80, and 90 min after in-fection, fixed for 1 hr in 10% Formalin, followed bythe standard Kellenberger and Ryter (6) osmium fixa-tion procedure, stained for 2 hr in uranyl acetate, de-hydrated through a graded acetone series, and em-bedded in Vestopal W. Sections were cut with dia-mond knives on an LKB Ultrotome, and stained with1% uranyl acetate in 60% methanol for 10 min andwith lead citrate by the method of Venable and Cog-geshall (13). Stained sections were examined in aHitachi Hu 11 electron microscope or in a Philips EM300 electron microscope.

RESULTS AND DiscussIoNThe host bacterium has the structural appear-

ance of a typical gram-negative rod (Fig. 1). Wehave been able to preserve only the outer mem-brane component of the cell wall [the L layer ofthe cell wall of Escherichia coli described by DePetris (1) ]. Although the host pseudomonadsappear as typical gram-negative rods, it has be-come increasingly apparent that these cells fre-quently contain an internal body which has theappearance of a myelin figure. This structure isseen in Fig. 2 and is quite similar to myelin figuresobserved by Kaye and Chapman (5) in colistin-treated E. coli, by Valentine and Chapman (12) inphage-infected C. marinoflava, and by Ryter andJacob in plasmolyzed cells of E. coli (8). Themyelin figures are most frequently found in thenucleoid region of the cell; however, they are oc-casionally observed at the periphery of the cell,as is clear in Fig. 3. Figure 4 is a particularlystriking demonstration of the myelin figure; onecan clearly detect that the periodicity betweeneach dark component of the myelin figure is quiteregular and measures approximately 25 A.The multiplicities of PM2 used to infect the host

bacterium in these experiments has not permittedus to observe in detail the initial stages of infec-tion, i.e., the attachment of the phage to the bac-terial cell. However, after 30 min one can see thatthe infected cells have suffered no structural al-teration. Figure 5 clearly shows that, after 30

min, there has been no change in the appearanceof the nucleoid region. Again, one can see myelinfigures in the cells; Fig. 6 demonstrates one myelinfigure at the periphery of a dividing cell. The Llayer of the cell wall can occasionally be seen tobleb away from the cell proper. Figure 7, whichis a micrograph of cells 45 min after infection,shows one such bleb within which one can ob-serve a myelin figure encased. It is of interest tonote that the myelin figure seen in Fig. 7 is actuallybetween the L layer and the cytoplasmic mem-brane. Such a location means either that themyelin figure has been extruded from the cyto-plasm, or that it never truly exists within thecytoplasm but that it normally is found in theperiplasmic space. However, if the myelin figurewere normally in the periplasmic space, one can-not easily understand why it can be found so fre-quently in the nucleoid region unless the peri-plasmic space actually is not limited to theperiphery of the cell but projects into the nucleoidregion. We have seen numerous examples of themyelin figure in the nucleoid region and in pro-truding blebs of the L layer. We hope to be ableto explain the structural relationship of themyelin figure within the cell by the more detailedultrastructural examination of the normal hostbacterium which is currently under way in ourlaboratory. The function of this structure is un-known; however, Ryter and Jacob (8) suggestedthat those myelin figures found in E. coli may bestorage deposits for lipids localized near the mem-brane.The bulk of the cells seen at 45 min after in-

fection appear quite normal (Fig. 7). However,an occasional cell has the appearance which wehave found to be characteristic for cells in thelater stages of infection with PM2. One such cellis seen in Fig. 8. This cell is highly distended, withan intact L layer and intact cytoplasmic mem-brane. The nucleoid is somewhat enlarged andthe cytoplasm is packed with ribosomes. Withinthe cytoplasm one can detect small but distinctparticles, of a diameter of 500 A, which lie closeto cytoplasmic membrane. These particles gen-erally reveal a densely staining central regionwhich is surrounded by a less densely stainingouter region. We believe that these particles areexamples of partially assembled or completedPM2 virions. It is clear that these particles arenot randomly distributed throughout the cyto-plasm but are always found at the periphery ofthe cell close to or adjacent to the cell membrane.The characteristic localization of the phage

particles in cells is quite clear 60 min after theonset of the infection (Fig. 9). A number of viralparticles can be seen in two of the three cellsdepicted. A tripartite envelope surrounds a

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COTA-ROBLES, ESPEJO, AND HAYWOOD

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FIG. 1. Thin section of normal, exponential-phase host pseudomonad. Only the L layer (L) of the cell wall hasbeen preserved. The bar in all electron micrographs represents 0.5 p. X 60,000.

FIG. 2. Normal cell ofhost bacterium containing myelin figure (MF) in nucleoid (N) region. X 60,000.FIG. 3. Normal cell of host pseudomonad containing myelin figure (MF) at the periphery of the cell. X 45,000.

58 J. VIROL.

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FIG. 4. Normal cell of host pseudomonad showing a well-resolved myelin figure in the nucleoid region. The lamel-lae contained in this myelin figure are approximately 25 A apart. This micrograph was taken with a Philips EM300 electron microscope. X 105,000.

59VOL. 2, 1968

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60 COTA-ROBLES, ESPEJO, AND HAYWOOD J. VIROL.

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FIG. 5. Host cells 30 miii after ouiset of inifectiont. Myelini figures (MF) canl be seeni in the niucleoid (N) regiont ofonie cell. X 60,000.

FIG. 6. Host cell 30 mili after intectionl showing myelin figure (MF) at periphery of cell. X 60,000.

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VOL. 2,1968 PM2, A LIPID-CONTAINING PHAGE 61

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FIG. 8. Host cell 45 min after infection. The cell appears swollen and deformed. PM2 particles (P) can be seen

at the periphery of the cell close to the cytoplasmic membrane. X 59,000.

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densely staining core in a number of the phageparticles, particularly the particle designated A.The viruses are close to the cytoplasmic mem-brane, and one in particular, labeled B, is virtuallyin apposition to the membrane. Cells at thisstage of infection are frequently extremelydistended, with a massive nucleoid region (Fig.10). One of the viral particles, labeled C, appearsto be attached to the cell membrane. This is madeclear (inset, Fig. 10) when the phage particle andmembrane are further enlarged. Our most carefulmeasurements cannot distinguish any differencebetween the thickness of the tripartite structurewhich surrounds the phage core and the thicknessof the cytoplasmic membrane. These measure-ments prove to be approximately 70 A.The fact that the virions are not distributed

randomly throughout the infected cell is under-scored in Fig. 11. At one pole of this cell, one cansee a striking array of six phage particles whichvirtually abut against one another and againstthe underlying cytoplasmic membrane. Con-tinued observation of virions adjacent to or incontact with the cytoplasmic membrane forces usto conclude that this localization is not fortuitousnor trivial. Such a precise association can be ex-plained by one of the two following alternatives:(i) the phage envelope is acquired from the cellmembrane of the host cell, i.e., the virus is coatedwith portions of the cytoplasmic membrane, or(ii) the phage envelope is synthesized de novo atthe membrane by the enzymatic apparatus whichsynthesizes cytoplasmic membrane. It is apparentthat the crude electron microscopic techniqueutilized in this investigation will not permit us toresolve this matter. Thus, one of us (R. T. E.) iscurrently investigating the source of the phos-phorus in the phospholipid of PM2. Preliminarystudies suggest that a considerable amount of thisphosphorus is derived from pools of phosphateextant in the cell prior to infection. One such poolcould of course be the phospholipid in the cyto-plasmic membrane. In any case, it is likely thatchemical or immunological means will permit theresolution of the question of phage localizationwhich has been made apparent by electron mi-croscopy.Some lysing cells are evident in our prepara-

tions of cells sampled 60 min after infection, whichsupports the turbidimetric studies (Espejo andCanelo, submitted for publication) made earlier.Figure 12 shows one infected cell in which the Llayer is greatly distended, although apparentlystill intact, whereas the cytoplasmic membrane isruptured. Some of the cytoplasmic contents havebeen released into the space delimited by the Llayer of the cell wall. Other lysing cells also appear

to suffer an initial degradation of the cytoplasmicmembrane prior to a rupture of the L layer. InFig. 13, the cytoplasmic membrane has been dis-rupted locally at point D. One bacteriophageparticle can be seen at the site of disruption of thecytoplasmic membrane. The extensive lysis whichhas occurred by 80 min is evident in Fig. 14. Inaddition to the numerous vesicles, one can see theremains of a cell (CF) in which virions are stillin close association to the membrane. Such an ob-servation suggests that the virions are not releasedimmediately upon lysis and that their associationwith the cytoplasmic membrane may be a realunion rather than a casual association. Furtherevidence for this interpretation is presented inFig. 15, in which one can see a large contortedcell fragment with a number of phage particlesstill in association with the cell membrane.The precise localization of PM2 with the cell

membrane of the host pseudomonad underscoresanother similarity between PM2 and animalviruses such as influenza virus and Newcastledisease virus. The presence of high levels of lipid,the sensitivity to organic solvents and detergents,the demonstration of an envelope in thin sections,and the site of viral completion offer tellingevidence in support of the view that PM2 mayindeed be a bacteriophage counterpart of certainanimal viruses. However, the absence of nucleo-protein from PM2 (Espejo and Canelo, submittedfor publication) emphasizes that PM2 cannot beconsidered an absolute counterpart of these ani-mal viruses.

It is conceivable that the unusual properties ofPM2 are the result of the rather unique selectionpressures exerted in its particular ecological niche,the marine environment. However, it is alsopossible that lipid-containing bacteriophages aremore widespread in nature but that they have notbeen observed; the tacit assumption that bac-teriophages are resistant to organic solvents mayhave made their discovery unlikely, since organicsolvents are frequently used to facilitate the isola-tion of bacteriophages. We well recognize that theisolation of a previously unexpected bacterium,Bdellovibrio (11), would not have been accom-plished if organic solvents had been used in theisolation of the plaque-forming entity.The isolation and characterization of a lipid-

containing bacteriophage is interesting in itself;however, further study of the relationship of thelipid envelope of PM2 to the cytoplasmic mem-brane of the host cell, as well as the study of thesynthesis of the lipid envelope, may contribute tothe understanding of the synthesis of such impor-tant macromolecular species as the phospholipo-proteins.

VOL. 2, 1968 63


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64 J. VIROL.

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65PM2, A LIPID-CONTAINING PHAGE

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66 COTA-ROBLES, ESPEJO, AND HAYWOOD J. VIROL.

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FIG. 13. Lysing cell 60 min after infection. The cytoplasmic membrane appears degraded at site D; the L layer(L) appears intact. X 65,000.

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VOL. 2,1968 PM2, A LIPID-CONTAINING PHAGE 67

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FIG. 15. Large cell fragment observed 90 miii after infection, showing numerous PM2 particles within the frag-ment. X 152,000.


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ACKNOWLEDGMENTS

This investigation was supported by Public HealthService grant A107460-01 from the National Instituteof Allergy and Infectious Diseases and by the Univer-sity of Chile-University of California CooperativeProgram.

Special thanks are due W. W. Thomson for per-mitting one of us (P. W. H.) to use the Philips EM300electron microscope.

LITERATURE CITED

1. DEPErR1S, S. 1967. Ultrastructure of the cellwall of Escherichia coli and chemical natureof its constituent layers. J. Ultrastruct. Res.19:45-83.

2. GARD, S., AND 0. MAAL0E. 1959. Inactivation ofviruses, p. 359-427. In F. M. Burnet and W.M. Stanley [ed.], The viruses, vol. I. AcademicPress, Inc., New York.

3. HOYLE, L. 1952. Structure of the influenza virus.J. Hyg. 50:229-245.

4. HOYLE, L. 1954. The release of influenza virusfrom the infected cell. J. Hyg. 52:180-188.

5. KAYE, J. J., AND G. B. CHAPMAN. 1963. Cyto-logical aspects of antimicrobial antibiosis.III. Cytologically distinguishable stages in anti-biotic action of colistin sulfate on Escherichiacoli. J. Bacteriol. 86:536-543.

6. KELLENBERGER, E., AND A. RYTER. 1958. Cell

wall and cytoplasmic membrane of Escherichiacoli. J. Biochem. Biophys. Cytol. 4:323-326.

7. MORGAN, C., R. A. RUKEND., AND H. M. ROSE.1962. The use of ferritin-conjugated antibodiesin electron microscopic studies of influenzaand vaccinia viruses. Cold Spring HarborSymp. Quant. Biol. 27:59-65.

8. RYTER, A., AND F. JACOB. 1966. Etude morpho-logique de la liason de noyau a la membranechez Escherichia coli et chez les protoplasts deBacillus subtilis. Ann. Inst. Pasteur 110:801-812.

9. SPENCER, R. 1963. Bacterial viruses in the sea,p. 360-365. In C. H. Oppenheimer [ed.],Symposium on marine microbiology. CharlesC Thomas, Publisher, Springfield, Ill.

10. STENT, G. 1963. Molecular biology of bacterialviruses. W. H. Freeman and Co., San Fran-cisco.

11. STOLP, H., AND M. P. STARR. 1963. Bdellovibriobactivorus gen. et sp. n., a predatory, ecto-parasitic and bacteriolytic microorganism.Antoine van Leeuwenhoek J. Microbiol.Serol. 29:217-248.

12. VALENTINE, A. F., AND G. B. CHAPMAN. 1966.Fine structure and host-virus relationship of amarine bacterium and its bacteriophage. J.Bacteriol. 92:1535-1554.

13. VENABLE, J. H., AND R. COGGESHALL. 1965. Asimplified lead citrate stain for use in electronmicroscopy. J. Cell. Biol. 25:407-408.

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