electron microscopic observations into gram-negative ... · into gram-negative bacterial hosts1...

JOURNAL OF BACTERIOLOGY, Oct. 1968, p. 1366-1381Copyright i 1968 American Society for Microbiology

Electron Microscopic Observations on thePenetration of Bdellovibrio bacteriovorus

into Gram-negative Bacterial Hosts1JEFFREY C. BURNHAM, T. HASHIMOTO, AND S. F. CONTI

Department of Microbiology, University ofKentucky, Lexington, Kentucky 40506

Received for publication 29 July 1968

The progressive stages in Bdellovibrio bacteriovorus penetration into two strains ofEscherichia coli were examined by use of electron microscopic techniques. Theinitial change observed in the ultrastructure of the host following parasitic attack wasthe swelling of the cell envelope at the site of attachment. The Bdellovibrio then ap-peared to pierce the center of this swelling, forming a pore in the outer wall layers ofthe host. The edges of this entry pore constricted the Bdellovibrio throughout itspenetration into the host cell. Although partial disruption of the cytoplasmic mem-brane was always apparent, the parasite did not appear to actively penetrate throughthis barrier. An attempt is made to correlate the fine structural changes involved inpenetration with the physiological data that have accumulated to date.

Ultrastructural investigations concerningBdellovibrio bacteriovorus have conclusivelyshown that this organism is an endoparasite; itis known to penetrate through the wall layers ofthe host bacterium and enlarge and lengthenwithin the confines of the host cell before dividinginto a variable number of progeny (3, 6, 9, 10).Scherff et al. (6) demonstrated that B. bacterio-vorus attacks the host cell (Pseudomonas fluores-cens) with the end of the cell opposite the sheathed(7) flagellum, using an anterior polar cushion toattach to and penetrate through the wall of thehost. In addition, they reported that the host cellafter infection assumed a spheroplast configura-tion; the cytoplasmic membrane was destroyedduring parasitic attack.

Starr and Baigent (9) described a similarparasitic interaction using three other gram-negative bacteria as hosts, Erwinia amylovora, P.phaseolicola, and P. tabaci. They showed that apore was formed in the host's cell wall throughwhich the parasite squeezed to enter the host cell.This was followed by a loss of the murein layerof the host cell wall with subsequent completeinternal disorganization of the host cell.

Similar observations were made with a B.bacteriovorus-Salmonella typhi system by Lepineet al. (3).

It was demonstrated (Abram and Shilo,Bacteriol. Proc., p. 41, 1967) that host-parasitecontact resulted either in the formation of blebs

1 A portion of this investigation was presented atthe Annual Meeting of the American Society forMicrobiology, Detroit, 1968.

or pits in the host cell wall. Several "rigid" fiberswere observed emerging from a "holdfast"structure present on the anterior end of Bdello-vibrio cells which might be involved in the attach-ment process. In addition, it was observed that ifmultiple attachment to a host cell occurred, thehost rapidly disintegrated without an apparentincrease in the number of parasites.

In view of the recent data of Varon and Shilo(14), it was decided to study further the penetra-tion process so that the progressive fine structuralevents of penetration could possibly be integratedwith the current physiological data.

MATERIALS AND METHODS

Organisms. B. bacteriovorus (strain 15143) wasobtained from the American Type Culture Collection.The host bacteria employed were two strains ofEscherichia coli: (i) strain UKS was isolated at theDepartment of Microbiology, University of Kentucky;(ii), E. coli B/r was obtained from George J. Hageage,Jr.

Medium. The medium employed in this investiga-tion was that previously described by Stolp and Starr(11) and designated YP. It consisted of 0.3%o (w/v)yeast extract and 0.06% (w/v) peptone (Difco), ad-justed to pH 7.6 with 1.0 N NaOH prior to autoclav-ing.

Cultural conditions. The host strains were culturedin 100-ml volumes of YP medium in 500-ml Erlen-meyer flasks on a rotary shaker maintained at 30 C.Routine procedures for maintenance of both thehost and the bdellovibrios have been described (9, 11).The host-parasite interaction was followed by harvest-ing 18-hr cultures of E. coli and B. bacteriovorus

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(cultures varied in age from 18 to 24 hr) by centrifuga-tion and mixing the bdellovibrios with an excess ofhost cells in an appropriate medium. The interactionwas observed with YP medium, distilled water, andthe supernatant fluid from a spent Bdellovibrio-E. coliculture medium; all suspending media were satis-factory. However, the bdellovibrios appeared to bemost voracious in their old culture supernatantfluid. Culture supematant liquid was obtained bycentrifuging out the bacteria and putting that super-natant liquid through a 0.22-,um membrane filter(Millipore Corp., Bedford, Mass.). For observationof unattached free-swimming bdellovibrios, an 18-hrmixed culture of B. bacteriovorus and E. coli wasfiltered through a 0.45-,um membrane filter (MilliporeCorp.), thereby eliminating the hosts. The parasiteswhen observed in this filtrate remained motile andretained their predaciousness if allowed to interactwith host bacteria.

Sampling procedure. Sampling was carried out atsuccessive time intervals determined by observationunder a phase contrast microscope of the stages ofparasitic attack and penetration. Samples were im-mediately mixed with glutaraldehyde [buffered atpH 6.1 with a modified veronal acetate buffer (2)] toyield a final aldehyde concentration of 1.0%. Thecells were then centrifuged and suspended in 6.25%glutaraldehyde in the modified veronal acetate buffer(2) for 12 to 24 hr at 4 C.

Electron microscopy. Following the initial glutaral-dehyde fixation, the samples were washed three timesin modified veronal acetate buffer and fixed for 12 hrin 2% osmium tetroxide dissolved in modified veronalacetate buffer at pH 6.1. Samples were suspended in0.5% aqueous uranyl acetate before being dehydratedin a graded acetone series. Samples were infiltratedwith a mixture of Epon 812 and 815 and polymerizedat 60 C for 24 hr.

Specimens were sectioned with a diamond knifeon an LKB Ultrotome and placed on 300- or 400-meshcopper grids (Ladd Research Industries, Burlington,Vt.). Serial sections were picked up on Formvar-coated special design copper grids (Ladd ResearchIndustries, 2524). All sections were postained withuranyl acetate and Reynolds' lead citrate stain (5).

Specimens were negatively stained with 2% (w/v)potassium phosphotungstate, adjusted to pH 7.1with 1 N KOH. Samples for electron microscopywere examined with a Philips EM 200 electronmicroscope operating at 60 kv.

RESULTS

When an active culture of B. bacteriovorus isallowed to attack an excess (2:1) of host or-ganisms, 75% attachment occurs in less than 15min as determined by phase contrast microscopy.A partial synchrony of the penetration processcan be obtained by this procedure.As has been observed by other investigators

(3, 6, 8, 14), the bdellovibrios actively andviolently strike the host organism with the end ofthe cell opposite the sheathed flagellum. Follow-ing an initial "recognition" period where attach-

ment is apparently reversible, the attachedbdellovibrios commence a gyrating motion, whenobserved by phase contrast microscopy, whichcontinues for a few seconds to several minutesbefore ceasing. Our observations indicate thatthe bdellovibrios retain their motility beyond thetermination of this gyratory motion; they occa-sionally push the host cells through the suspend-ing medium.

Attempts to separate the parasite from thehost cell during the early stages of attachmentwere unsuccessful. Neither violent shaking normixing the cells in a Vortex Genie (ScientificInstrument Co., Springfield, Mass.) at maximalrotation had any visible effect. Brief sonic treat-ment did not separate them and longer periods ofsonic treatment resulted in partial disruption ofhost and parasite cells. These initial experimentssupport the previous reports of irreversible con-tact and strong bonding between the parasite andthe host bacterium (9, 11, 13).When unattached bdellovibrios were examined

by electron microscopy, several features wereapparent which differentiate the organism fromother typical gram-negative bacteria.

Figure 1 is a negatively stained preparation,showing the convoluted surface of the bacteriumand the thick polar flagellum with its dampedwavelength.The anterior end of some unattached Bdello-

vibrio cells (i.e., that opposite the flagellated end)appeared distorted by large convolutions whichappear in negatively stained preparations (Fig. 1and 2) to be part of the cell wall itself. Thesestructures (Abram and Shilo, Bacteriol. Proc.p. 41, 1967) in negatively stained preparationsformed a "holdfast." It has been suggested thatsuch a structure has a functional role in cellpenetration (6). However, we have found noevidence to support or negate this contention.

Figure 3 illustrates the appearance of consider-ably longer, spiral-shapedBdellovibrio cells whichare occasionally present in young parasitic cul-tures. These long forms increase in number inolder cultures; we observed that these forms canattach to host cells.When the bdellovibrios were examined in thin

sections, apparent "holdfasts" were often presentand appear as exaggerated convolutions or serra-tions of the outer cell wall (Fig. 4). All micro-graphs obtained of this anterior region to dateshow no distinct stainable material occupyingthe enlarged space between the distorted outercell wall layers and the cytoplasmic membrane.These observations suggest that this region maybe more susceptible than the rest of the cell todistortion by the methods of preparing specimensfor electron microscopic examination.

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1368 BURNHAM, HASHIMOTO, AND CONTI J. BACTERIOL.

FIG. 1. B. bacteriovorus ATCC 15143 negatively stained, showing the vibrio-shaped cell with its thick polarflagellum and the large surface convolutions on the anterior end of the cell (arrow).

FJG. 2. B. bacteriovorus negatively stained illustrating the distended anterior tip or "holdfast" (arrow) of theparasite.

FIG. 3. Negatively stained preparation ofa B. bacteriovorus "lonig" form; this type of cell occasionally appearsin 18-hr cultures but becomes more prevalent in older cultures.

FIG. 4. Thin section of unattached Bdellovibrio, illustrating the dense cytoplasm and densely staining bodies(DB) occupying part of the chromatin area. The anterior end of the cell contains a lamellar mesosome (M) anda distended outer cell wall forming the "holdfast" (arrow) shown in Fig. I and 2.

FIG. 5. A thin section ofB. bacteriovorus showing the polar flagellum to be sheathed (arrow) by a continuationof the cell wall.

FIG. 6. A thin section ofE. coli B/r illustratintg its cytoplasm, ribosomes (R), the densely staining area (peri-plasm, P) between the outer cell wall (OW) anid the cytoplasmic membrane (CM).

FIG. 7. Thin section shows a slightly plasmolysed cell oJ E. coli B/r. The cell wall (OW) is 0.75 pm wide, theperiplasmic layer (P) is 1.3 pm wide, and the cytoplasmic membrane (CM) is 0.75 pm wide.

FIG. 8. An earlv stage of Bdellovibrio penetration into E. coli. The parasite is inivaginating the bulge (arrow)formed by the cell wall of the host and breaking apart the outer layers of the cell wall. The small bleb (B) at theattachment site is formed by protruding outer layers of the cell wall.

FIG. 9. Thin section of B. bacteriovoruts breakinig apart the outer layers of the host's cell wall (arrow). Otherareas of the host cell appear normal, except for the spaces (double arrow) at the corners of the bulge formation.

FIG. 10. Serial sections showing two Bdellovibrio cells simultaneously attacking ani E. coli UKS.

FIG. 11. A thin section of Bdellovibrio penetration into E. coli UKS, showing the widening of the penetrationpore (arrows) and presence of blebs located at inicreasing distances from the pore site.

FIG. 12a,b. Thin-section o, B. bacteriovorus penetrating into an E. coli UKS, showing the formation of a "pit"in the cell wall of the host at the site ofpenetration.

FIG. 13. Thin-section of B. bacteriovorus during the middle stages of penetration, showing the constriction(arrow) of the parasite by the cell wall layers (O W) of the host. The Bdellovibrio is invaginating the membrane ofthe host. Note the loss of the dense periplasmic layer (P) beneath the outer wall layers.

FIG. 14. Serial sections of B. bacteriovorus penetrating into E. coli UKS, showing in particular the constrictionof the parasite by the penetration pore (arrows) in the host cell, the bulge in the host cell wall, and the partiallydeteriorated periplasmic layer.

FIG. 15. A thin section showing a Bdellovibrio cell near completion of penetration into an E. coli B/r. Con-striction is still evident (arrow) at this late stage ofpenetration.

FIG. 16. B. bacteriovorus in a late stage ofpenetration. The parasite has not penetrated the cytoplasmic mem-brane (CM) of the host but instead occupies the space between the outer cell wall and the cytoplasmic membrane.

FIG. 17. A thin section showing E. coli UKS containing a Bdellovibrio withinz a membratne invagination.FIG. 18. Thin section ofE. coli B/r with a Bdellovibrio located within the cytoplasm of the host. The host cell

shows no evidence of being converted to a spheroplast.


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In addition to the above structure, in most ofthe bdellovibrios observed in thin sections, amesosome comprised of a variable number oflamellar membranes was closely associated withthe anterior end of the cell (Fig. 4, 10, 13, 14).

Figure 5 illustrates that the flagellum of B.bacteriovorus is surrounded by a sheath con-tinuous with the cell wall. The diameter of theflagellum, including the sheath, is 28 nm; thisvalue is similar to that reported by Seidler andStarr (6).A thin section of the E. coli B/r (Fig. 6) shows

the general ultrastructural features of this gram-negative bacterium. The cell envelope consists ofa cell wall 0.75 pm wide and a cytoplasmic mem-brane of similar dimensions. The rigid or "R"layer observed in electron micrographs of manygram-negative bacteria as a thin dense line (1, 4)was missing from our preparations. Instead a1.2- to 1.5-pm thick electron-dense matrix (peri-plasmic layer) occupied the space between thecytoplasmic membrane and the cell wall. Anenlargement of the cell wall of a slightly plas-molysed E. coli can be observed in Fig. 7. Thedense periplasmic layer separated from the cyto-plasmic membrane and adhered to the inner sur-

face of the cell wall.The first step in penetration involved the at-

tachment of the parasite to the host cell wall andresulted in a flattening of the outer membranein the "holdfast" region. Following this initialattachment, the first observable change in the hostcell ultrastructure was a bulging of the wall andmembrane at the point of attachment (Fig. 8 and9). Figure 8 shows the Bdellovibrio apparentlypushing into the center of the bulge. Fig. 9, ahigh magnification micrograph of the region ofbulge formation in the cell wall of the host,illustrates the Bdellovibrio breaking through thecell wall of the host. The cytoplasm of the hostat this point was not disrupted, although slightseparation of the cytoplasmic membrane fromthe outer layer of the cell wall can be noticed atthe edges of the swelling.

Figure 10 illustrates the value of serial section-ing in the interpretation of the penetration proc-ess. The four serial sections reconstruct a smalland medium-sized bulge formed by two bdello-vibrios in an early stage of penetration and themesosome in the anterior tip of one of the para-sites.

Figure 11 shows the widening of the penetra-tion pore in the cell wall and the marked effectsof parasite penetration through the cell wall ofthe host as evidenced by the formation of blebslocated at increasing distances from the penetra-tion pore.

A few of the bdellovibrios observed in variousstages of penetration produced a unique "pit"formation in the host cell (Fig. 12). The mem-branes within the "pit" appear in serial sectionsto be disrupted, thereby forming spherules andtubules. The reason for "pit" formation, and thefunction, if any, of these structures, are unknownto us at this time.The violence of parasitism that is suggested by

phase contrast observations is structurally repre-sented in Fig. 13. The host cell wall is severelydisrupted carrying along with it a portion of thecytoplasmic membrane. In addition, the host cellwall appears to constrict the Bdellovibrio at itspoint of entry into the host. At this point in theparasitic process, a gradual deterioration in theperiplasmic matrix of the host cell occurs at thesite of penetration; this progressive deteriorationsubsequently is observed in all areas of the peri-plasm. Although the cytoplasmic membrane isobserved to be broken in places, the Bdellovibriodoes not appear to be breaking through it; themembrane seems to be pushed inward by theparasite. The effect of hypotonic medium pene-trating into the host spheroplast apparently ismanifested by a dilution of the host's cytoplasm.

Figure 14 again represents the value of serialsectioning in the interpretation of host-parasitecell interaction. Serial sectioning allows a three-dimensional reconstruction of the penetrationsite, and in this instance, shows the constrictionof the Bdellovibrio cell as it squeezes through thepore in the cell wall of the host. In addition, anapparent loss of the electron dense material adja-cent to the outer layers of the cell wall can be ob-served, indicating that the periplasmic region hasbeen affected.

Figure 15 illustrates one of the final steps of theparasite's penetration into the host cell. TheBdellovibrio is still being constricted by the outerlayers of the host's cell wall, and very often sec-tions show that the Bdellovibrio completes itsinvasion without actively penetrating into thecytoplasmic membrane of the host cell (Fig. 16).

Figure 17 shows the Bdellovibrio, completelywithin the host, occupying an invagination of thecytoplasmic membrane that accounts for theapparent presence of a double membrane. Theparasite does not necessarily occupy the spacebetween the membrane and the cell wall in thehost cell (Fig. 18), presumably because whenexternal forces are great enough, the membraneis so disrupted that there is no barrier for pene-tration by the parasite. In these instances, thecytoplasm of the host cell is usually very dis-organized.

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B. BACTERIOVORUS PENETRATION INTO HOST BACTERIA

FIG. 19. Diagrams representing the penetration ofB. bacteriovorus into E. coli. Diagram I represents the earlieststage ofattachment, showing no effect ofattack on the cell wall of the host. Shown are the cell wall (O W), a middledense layer (P), the cytoplasmic membrane (CM) of both the host and the Bdellovibrio, mesosome (M), blebs(B), and spherules (S). This sequence is constructed on the basis ofour light and electron microscope observations.

DIscussIoNThe various phases of B. bacteriovorus penetra-

tion into host bacteria are pictorially presentedin Fig. 19. Bulge formation is shown as the firstobserved structural change in the host. This isfollowed by breaching of the outer cell wall layerby the anterior tip of the parasite.The last four diagrams present the final stages

of Bdellovibrio penetration, illustrating in par-ticular the continual constriction of the parasiteby the rigid outer layers of the host cell wall. Inaddition, the characteristic blebs (Fig. 19e)

formed by the host cell wall are shown, and thedense periplasm (Fig. 19a) is shown to deteriorategradually until little residue remains upon com-pletion of penetration. The penetration pore is notrepresented in the last diagram (Fig. 19h) be-cause we have not determined whether the poreis sealed off or remains open after the Bdellovibriohas completely entered the host.The diagrams and the micrographs presented

illustrate stages of parasite attachment and pene-tration. Membrane decomposition, parasiteelongation and division, and the loss of host

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cytoplasm that all occur in later stages of parasit-ism are not represented.The violent collision of the parasite with the

host and the subsequent rotating motion havebeen hypothesized (10, 12) to be responsible forthe pore formation in the host cell wall throughwhich the parasite penetrates. Our investigationsuggests that the initial damage to the host cellis the formation of a bulge in the cell wall (Fig.19b, c). Following this, the outer membrane isbroken (Fig. 19d); the rigidity and appearance ofthe edges of the outer cell wall adjacent to theentry pore suggest that these are physical phe-nomenona and are not caused by enzyme action.Our micrographs indicate that the constriction ofthe Bdellovibrio is due only to the outer wall ofthe host (Fig. 13) because often no other wallstructures are observable. Our current view isthat the structure responsible for wall rigidity isnot observable and has not yet been removed bythe enzymes of the Bdellovibrio, whereas the restof the periplasm has been destroyed, perhaps byaction of the proteases previously described (8).Further evidence supporting this hypothesis is theabsence of host spheroplasts during the earlystages of the parasitic process. Spheroplasts be-come prevalent during the middle stages of para-sitic attack and occasionally have not been formedafter penetration has been completed (Fig. 18).Our results, in agreement with those of Starr

and Baigent (9), suggest enzymatic decomposi-tion of the periplasmic matrix, although it is notapparent whether this is necessary for penetrationto occur. It is known (14) that some enzymes areinvolved, but their site of action is not known. Itis possible that enzymes from the Bdellovibrio(possibly associated with the anterior mesosome)serve to release the attachment bonds of theparasite to the host allowing the Bdellovibrio toproceed into the host cell without drawing in thelayers of the host wall with it.Varon and Shilo (14) indicated that motility

is necessary for attachment to occur. It wouldappear from our micrographs that motility mightbe needed during penetration to supply also theforce necessary to counteract the constant pres-sure exerted on the Bdellovibrio by the edges ofthe pore in the host cell wall.Our current hypothesis of Bdellovibrio pene-

tration is constructed with the above observationsin mind. Following the recognition phase whereattachment is reversible, a tight bonding of theBdellovibrio to the outer layers of the host cellwall occurs. The apparent drilling action doesnot appear to involve a spinning along the axisof the parasite but a swivelling of the posteriorend of the parasite with no rotation of the attach-ment tip. This violent action would have the

effect, when the Bdellovibrio is coupled tightly tothe host, of weakening this region of the host'scell wall, thus rendering this area of the wallsusceptible to osmotic forces, possibly producingthe observed swelling.

It is conceivable that following bonding of theparasite to the host and initial bulge formation,the physical breaking of the host's cell wall isaccomplished by a centripetal growing pointlocated at the center of the attachment tip. Thishypothesis involves the production by theBdellovibrio, possibly in association with thepolarly oriented mesosome, of new cell wallmaterial, which evaginates and forces the cellwall of the host to part.The gradual release of the parasite's attach-

ment bonds to the cell wall of the host could beaccomplished by lytic enzymes produced by theBdellovibrio. This would allow the parasite tocomplete its entry, through the penetration pore,into the host cell.

AcNowLEDGmxNTsWe acknowledge the technical assistance of Bruce

Tutein throughout the course of this study.This investigation was supported by grant GB

5336 from the National Science Foundation.

LrERATuRE CITED1. DePetris, S. 1967. Ultrastructure of the cell wall

of Escherichia coli and chemical nature of itsconstituent layers. J. Ultrastruct. Res. 19:45-83.

2. Kellenberger, E., A. Ryter, and J. S6chaud. 1958.Electron microscope study of DNA-containingplasms. II. Vegetative and mature phage DNAas compared with normal bacterial nucleoids indifferent physiological states. J. Biophys.Biochem. Cytol. 4:671-678.

3. Lepine, R. M., A. Guelin, J. Sisman, and D.Lamblin. 1967. Etude au microscope electro-nique de la lyse de Salmonella par Bdellovibriobacteriovorus. Compt. Rend. 264:2957-2960.

4. Murray, R. G. E., P. Steed, and H. E. Elson. 1965.The location of the mucopeptide in sections ofthe cell wall of Escherichia coli and othergram-negative bacteria. Can. J. Microbiol.11:547-560.

5. Reynolds, E. S. 1963. The use of lead citrate athigh pH as an electron opaque stain in electronmicroscopy. J. Cell Biol. 17:208-212.

6. Scherff, R. H., J. E. DeVay, and T. W. Carroll.1966. Ultrastructure of host-parasite relation-ships involving reproduction of Bdellovibriobacteriovorus in host bacteria. Phytopathology56:627-632.

7. Seidler, R. J., and M. P. Starr. 1968. Structure ofthe flagellum of Bdellovibrio bacteriovorus.J. Bacteriol. 95:1952-1955.

8. Shilo, M., and B. Bruff. 1965. Lysis of gram-negative bacteria by host-independent ecto-parasitic Bdellovibrio bacteriovorus isolates. J.Gen. Microbiol. 40:317-328.

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9. Starr, M. P., and N. L. Baigent. 1966. Parasitict interaction of Bdellovibrio bacteriovorus with

other bacteria. J. Bacteriol. 91:2006-2017.10. Stolp, H. 1968. Bdellovibrio bacteriovorus-ein

riiuberischer Bakterienparasit. Naturwissenscha-ften 55 :57-63.

11. Stolp, H., and M. P. Starr. 1963. Bdellovibriobacteriovorus gen. et sp. n., a predatory ecto-parasitic, and bacteriolytic microorganism.Antonie van Leeuwenhoek J. Microbiol.Serol. 29:217-248.

12. Stolp, H., and M. P. Starr. 1965. Bacteriolysis.Ann. Rev. Microbiol. 19:79-104.

13. Stolp, H., and H. Petzold. 1962. Untersuchungenuber einen obligat parasitischen Microor-ganismus mit lytischer Activitiit fur Pseudo-monas-Bakterien. Phytopathol. Z. 45:364-390.

14. Varon, M., and M. Shilo. 1968. Interaction ofBdellovibrio bacteriovorus and host bacteria.I. Kinetic studies of attachment and invasionof Escherichia coli B by Bdellovibrio bacterio-vorus. J. Bacteriol. 95:744-753.

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electron microscopic observations into gram-negative ... · into gram-negative bacterial hosts1...

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