Bacillus mycoides phages and their satellites

ANNA S. TIKHONENKO, NINA N. BELYAEVA, AND ANNA F. KRETOVA Institute of Molecular Biology, U.S.S.R. Academy of Sciences, Moscow, U.S.S.R.

Accepted January 30, 1984

TIKHONENKO, A. S., N. N. BELYAEVA, and A. F. KRETOVA. 1984. Bacillus mycoides phages and their satellites. Can. J. Microbiol. 30: 69 1 -698.

The relationship between large and small particles of phages No. IM and H17 reproducing simultaneously in one and the same bacterial cell of Bacillus mycoides was studied by the immune electron microscopic technique. The large particles of phages No. 1M and H17 were morphologically identical with phage NO. 1 of B. mycoides, whereas only the tails of small particles of phages No. 1M and H17 were morphologically identical with the tail of phage No. 1. Antigens were identified in phages No. 1, No. lM, and HI7 using specific antibodies against phage No. I, containing only large phage particles, and specific antibodies against phage H17 small heads. It was shown that ( i ) all structural elements of large particles and tails of small particles of phage No. 1M were antigenically identical with those of phage No. 1; (ii) all structural elements of small and large particles of phage H17, except the inner core of the tail, were antigenically different from phage No. 1; and ( i i i ) the small heads of phages No. 1M and HI7 were antigenically identical. Particles of phage No. 1 are morphologically and antigenically identical with the large particles of phage No. 1M and are antigenically different from the large particles of phage H17. Since the tails of small and large particles are antigenically identical in each phage pair (No. 1M and H17), this suggests that in both cases, the genome of a small defective phage codes for the synthesis of head proteins only, whereas its tail is borrowed from the corresponding helper phage. The small phage may therefore be considered as a satellite of the large phage which depends on a helper partner for production of complete particles and whose tail proteins are identical with those of the helper phage.

TIKHONENKO, A. S. , N. N. BELYAEVA et A. F. KRETOVA. 1984. Bacillus mycoides phages and their satellites. Can. J. Microbiol. 30: 691 -698.

La parent6 entre grandes et petites particules des phages No. 1M et H17, propagCs simultantment dans le m&me cellule de Bacillus mycoides, a t t t ttudiCe par microscopic Clectronique. Les grandes particules des phages No. 1M et H17 sont morphologiquement identiques avec celles du phage No. 1 de B. mycoides. Chez les petites particules des phages No. 1M et H17, seules les queues sont identiques avec celle du phage No. 1. La structure antigtnique des phages No. 1, No. 1M et H17 a CtC CtudiCe ?I l'aide d'anticorps spCcifiques contre le phage No. 1 produisant seulement des grandes particules et contre les t2tes petites du phage H17. Les rtsultats dtmontrent que ( i ) tous les ClCments structuraux des grandes particules ainsi que les queues des petites particules du phage No. 1M sont antigeniquement identiques avec les structures correspondantes du phage No. 1, ( i i ) toutes les structures des grandes et petites particules du phage H17, ?I l'exception du stylet axial de la queue, sont antigtniquement diffkrentes de celles du phage No. 1, et (iii) les petites t&tes des phages No. 1M et H17 sont antigeniquement identiques. Les particules du phage No. 1 sont morphologiquement et antigkniquement identiques aux grandes particules du phage No. 1M et diffkrent antigeniquement des grandes particules du phage H17. L'identitC antigknique des queues phagiques des grandes et des petites particules ainsi que chez le phage NO. 1M et H17 suggkre que dans les deux cas, le gCnome d'un petit phage dtfectif code pour la synthkse des protCines de la t2te seulement tandis que la queue est empruntCe au phage auxiliaire correspondant. On peut donc considCrer le petit phage comme un satellite du grand virus et dCpendant d'un phage auxiliaire pour la production de particules compl&tes et ayant les m&mes protCines de queue que celui-ci.

[Traduit par le journal]

Introduction As has been reported in the literature by several

authors, a phage lysate can contain two forms of phage particles having heads of different size and tails identi- cal in size and structure (Tikhonenko 1970; Tikhonenko and Bespalova 1961; Shore et al. 1978). Such phages cannot be separated by conventional biological tech- niques. The nature of this phenomenon has been eluci- dated only for Escherichia coli K-235 phages P2 and P 4 (Shore et al. 1978). The genetic and electron micro- scopic studies of the structural and functional rela- tionships between these two phages have shown that satellite phage P4 requires all the morphogenetic gene products provided by the helper phage P2 to assemble

its own capsid, which is one-third the volume of the larger helper capsid. Phage P4 takes proteins for tail formation from phage P2.

The phenomenon of simultaneous reproduction of two phage forms in the same bacterial cell was reported earlier for Bacillus mycoides strain No. 1 (Tikhonenko and Bespalova 196 1).

W e isolated phages No. 1 M and H17 from soil and studied them by electron microscopy. These plaques simultaneously contain phage particles with large and small heads and tails of identical structure and size. Phage particles with a large head are identical in their morphology and size with the earlier described B . my- coides phage No. 1, containing only large phage par-

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FIG. 1 . Bacillus mycoides phage No. 1 . FIG. 2. (a) Bacillus mycoides No. 1M and ( b ) phage H17. Bars = 100 nm.

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TIKHONENKO ET AL. 693

TABLE 1. Morphology of phages No. 1, No. IM, and H17

Width of extended phage tail

Head Tail Proximal Distal Width of diameter length vart part contracted

Phages (nm) (nm) (nm) (nm) tail (nm)

Phage No. 1 8 1 200 14 17 20

Phage No. 1M Large 81 200 14 17 20 Small 30 200 14 17 20

Phage

Large 8 1 200 14 17 20 Small 30 200 14 17 20

ticles (Tikhonenko 1961). The present work was aimed at studying the re-

lationship between large and small phage particles growing simultaneously in one and the same bacterial cell by identifying antigens in B. mycoides phages No. 1 , No. l M , and H17.

Materials and methods Bacteriophages

: Three B . mycoides phages (No. 1, No. lM, and H17) were used in the work. The phages were isolated from different soil

: samples using the enrichment procedure. The atypical B. my- coides strain No. 1 grown on Difco nutrient broth and nutrient agar at 27°C was used as a host and test culture. Plaques were obtained by the agar bilayer method of Adams (1959).

Concentrated phage suspensions were obtained from plates with confluent lysis according to Adams (1959), purified by differential centrifugation and dialysis against 0.14 M NaCl

. . . . . solution.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . :: - : . ... :. .:.:..:.:...::.:.:. . . . . Small phage heads were isolated by ultracentrifugation at . . . . . . . . . . . . . . . . . . . . . . . . . . .: - . . - .: :. .__ .; _._ _, . . . : . ,. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : .: 20000 rpm for 3 h (Spinco ultracentrifuge with an SW 40 Ti

. . . . . bucket rotor, Beckman) in a CsCl step gradient buoyant den-

. . . . . . . . . .. . . 1 . _v . . . . . . . . . . . . . . . . . . . . . .

. . sities of 1.2 and 1.4 g/mL. . . . . . . . . . . . .

. . Rabbit antisera against phage No. 1 and the small heads of

phage H17 were prepared by injecting the antigens and an adjuvant intramuscularly.

Antibodies were purified by fractionation of immune sera on DEAE-cellulose: 1 mL of the antiserum was loaded onto a small column (volume = 5 mL) of DEAE-cellulose and was equilibrated with 0.0175 M phosphate buffer (pH 6.3). The same buffer was used for elution. Fractions (2 mL) were collected and their optical densities were measured on a spec- trophotometer (SF-26, "LOMO," U.S.S.R.) at 280 nm. The collected fractions were examined by the electron microscope for antigen-antibody complex formation.

Immunoelectron microscopy The antigen-antibody reaction was performed directly on

a grid canying a holey carbon film supported by a thin film

of Parlodion. One drop of phage suspension (about 10" particles/mL) was added to the grid and after about 30 s it was washed off with a drop of distilled water. Then the grid was floated with the specimen side down on an antibody solution in a small plastic tube. The grid was incubated with the antibodies at 37OC for 30 min. It was then washed with three drops of distilled water and negatively stained by a drop of 4% aqueous solution of sodium silicotungstate at pH 7 .0 for 30 s. ~ x c e s s fluid was removed with filter paper. Specimens were observed with a JEM lOOB electron microscope at 80 kV with a 50 000 X magnification on the screen.

Calibration of magnification of the electron microscope was achieved using a diffraction grating with 2050 lines per 1 Fm.

The sizes of phage particles were established by measuring 100 phage particles to obtain an average value. The diameters of heads were obtained by measuring the distances between opposite apices.

Results and discussion Bacillus mycoides phage No. 1 forms transparent

plaques with a diameter up to 3-5 mm in which only phage particles with large heads can be discerned.

Plaques of phages No. 1M and H 17 have a diameter up to 10 mm with alternating turbid and transparent zones. Electron microscopic examination of the ma- terial taken from the plaques of phages No. 1 M and H17 showed that they contained simultaneously two forms of phage particles, with large and small heads, while their tails were morphologically identical.

Numerous successive passages of the material taken from single plaques did not make it possible to separate small and large phage particles. Moreover, as was shown earlier, phage No. 1M particles with small and large heads reproduced simultaneously in the same bacterial cell (Tikhonenko and Bespalova 1961). Large particles of phages No. 1M and H17 are entirely identi- cal, in their morphology and size, with B. mycoides phage No. 1 studied by one of us (Tikhonenko 1961).

As shown earlier (Tikhonenko 1961), the intact No. 1 phage particle consists of a head 81 nm in dia- meter, whose capsid is composed of capsomers, and a tail 200 nm in length (Fig. 1 ) . The tail is a complex structure and consists of an outer sheath and an inner hollow core. The sheath is made up of easily discernible morphological subunits and terminated with a base plate with six prongs. A single thin fibre extends from the centre of the base plate. The inner hollow core is exposed on contracted tails.

The head diameter of large particles in phages H17 and No. 1M is also about 81 nm, whereas the head diameter of small phage particles is 30 nm.

Phages No. 1, H17, and No. 1M are indistinguish- able in morphology and size of their tails (Figs. 2a, 2b; Table 1 ) .

The electron microscopic study.of phages No. 1M

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FIG. 3. Interaction of phage No. 1 with homologous antibodies: ( a ) intact particles and ( b ) a particle with the contracted sheath. Bars = 100 nm.

and H17 has revealed a difference in the proportion of predominantly small particles, in particular many small phage particles with large heads and those with small heads with a neck but without tails (Fig. 2a, 2b). heads. Phage No. 1M contains much fewer small par- The relationship between large and small phages was

, ticles than large ones; in contrast, phage H17 contains studied using the technique of immune electron micros-

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TlKHONENKO ET AL.

FIG. 4. Interaction of phage No. 1M with antibodies against phage No. 1. The antibodies are attached to the tail of large and small phage particles as well as to the large phage head. The small phage head is free of the antibodies. FIG. 5. Treatment of intact phage H17 particles with antibodies against phage No. 1. The tail as well as the large and small phage heads are free of the antibodies. FIG. 6. Antibodies against phage No. 1 are attached only to the naked inner cores of the contracted tails of large and small phage H17 particles. FIG. 7 . Adsorption of antibodies against phage No. 1 on a polycore of phage H17 (arrows). Bars = 100 nrn.

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TABLE 2. Reactions of B. mycoides phages NO. 1, No. lM, and H17 with antibodies

Phage No. l Phage No. IM Phage HI7

copy for identification of antigens. Interaction of phage HI 7 with antibodies against phage To this end, antibodies were prepared against phage No. I

No. 1 and small heads of phage H17 to study the Figure 5 shows intact particles H17 after contact with immune complexes formed between specific antibodies antibodies against phage No. 1. One can see that neither and the phages. large nor small phages form specific complexes with

Antihdie a q a t phage No. I

Anlibodies against heads of small phage HI7

Interaction of phage No. IM with antibodies against phage No. I

The specificity of the isolated antibodies against phage No. 1 was tested by studying the formation of complexes between phage No. 1 and homologous anti- bodies. As can be seen from Fig. 3, the antibodies react in a specific manner with all the protein components of phage No. 1, since the head, the tail, the base plate, and the core of phage No. 1 exposed after the sheath con- traction are surrounded with a dense, 12 nm thick layer of the antibodies. No complexes are formed when the serum from a nonimmunized rabbit is used.

Specific complexes are also formed when phage No. 1M is treated with antibodies against phage No. 1. Whereas the antibodies reacted with all structural ele- ments of the large phage particles, they formed a complex solely with the tail components of the small particles. The capsid proteins of the head and neck of the small phage did not react with the antibodies against phage No. 1 (Fig. 4).

It follows that ( i ) the phage No. 1 and large No. 1M particles are identical antigenically; ( i i ) tail proteins of phage No. 1 and tail proteins of large and small par- ticles of No. 1M phage are morphologically and sero- logically related; ( i i i ) capsid proteins of small No. 1M particles are antigenically different from those of the large heads of phage No. 1 and large heads of No. 1M particles (see Table 2). Hence, the capsids of large and small heads of phage No. 1M consist of different

antibodies against phage No. 1: When phage H17 par- ticles with the contracted tail sheaths are treated with antibodies, the specific complex i s formed only on naked tail cores of the large and small phage H17 particles (Fig. 6).

It is noteworthy that defective phage structures (poly- cores) are found in H17 preparations. These structures also adsorb specific antibodies against phage No. 1 (Fig. 7). The relatively loose arrangement of the anti- bodies on these structures may be attributed to con- formational changes in the proteins of polycores as compared with the normal structure of tail cores. Ap- parently, certain antigenic determinants are masked during formation of polycores. A similar phenomenon has been observed in the serological study of polyheads of T-even phages (Yanagida et al. 1970).

The serological investigation of phage H17 has shown therefore that nearly all the structural elements of large and small particles (with the exception of tail cores) are different from phage No. 1 antigenically (see Table 2). One may suppose that the large phage H17 and phage No. 1 are two different phages regardless of their morphological similarity, since the large phage H17 differs from phage No. 1 in the antigenic nature of proteins, with the exception of tail core proteins.

Since large and small phages H17 are identical in the structure of their tails as well as in their antigenic char- acteristics, the small variety of H 17 may also be consid- ered as a satellite of the large phage H17.

0

proteins, while thkir tails are made up of antigenically Separation of large and small phage HI 7 particles and identical proteins. One may suppose that the small infection of B . mycoides cells by small phage phage is a satellite of the large phage and takes its particles proteins for the assembly of the tail from the helper To find out whether the small phage is propagated phage, i.e., from the large phage No. 1M. independently in the cell and can form complete par-

Q 0

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FIG. 8a. Small phage H17 after ultracentrifugation in a CsCl step gradient. FIG. 8b. Small H17 heads without tails are produced after infection of the bacterial cell with small phage H17 particles only. FIG. 9a. The antibodies against phage H17 small heads are adsorbed only on the heads of the small phage. FIG. 9b. The large phage head is free of antibodies against phage H17 small heads. FIG. 10. The antibodies against phage H17 small heads are adsorbed only on the small heads of phage No. 1M particles. The large phage head, as well as the tails of large and small phage particles, are free of antibodies. Bars = 100 nm.

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CAN. 1. MICROBIOL. VOL. 30. 1984

ticles, we had to separate it from the large particles and to infect the cell solely with the small phage particles. This separation was carried out by ultracentrifugation in a CsCl step gradient with buoyant densities of 1.2 and 1.4 g/mL which yielded a zone containing merely small phage particles (Fig. 8a). When the B . mycoides cells were infected with small phage particles, the phage was found to be capable of infection, but not able to lyse the cells; here, only small phage heads with necks but without tails were produced (Fig. 8b). Com- plete small phage particles with a head and a tail appear only when the cell is infected with large and small phages simultaneously. This supports our hypothesis that the small phage takes its tail from the large one.

. . . . It must be noted that satellite particles of B . mycoides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . .

phage are morphologically identical to many "killer- . . . . . . . . ,

. . . . particles" of Bacillus. But in our case these satellite . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . phages are not killers, since they can be arranged into

. . . . . . . . . . . . . . . . . . . . . . . . . ~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . :.. ........... :: ::...:..:..:.. ;. :: ..: .:.:.I a complete particle at the expense of phage helper. Then .......... . . . . (. .. :,. . . . . . . . . . . . _ . . . . . . , . . . . the satellite particles acquire an ability to infect the cell

with its genetic material which is capable of providing a reproduction of small heads only.

j Interaction of phages No. IM and HI 7 with antibodies

I against small heads of phage H17

A further objective was to study the antigenic identity of small heads in phages No. 1M and H17. To this end, we used specific antibodies against small heads of phage H17 (Fig. 8b). When phage H17 was treated with these antibodies, antigen-antibody complexes were found only on small phage heads. Large phage heads were free of antibodies (Fig. 9). Consequently, the proteins of large phage heads differ qualitatively in their antigenic properties from those of small heads.

We studied the interaction of antibodies against phage H17 small heads with phage No. 1M particles

and found that the small heads were covered with a thick layer of antibodies, while the large heads were free of them (Fig. 10). This is indicative of the fact that small head proteins of phages No. 1M and H17 share antigenic determinants (see Table 2). An antigenic relationship thus exists between the head proteins of the small phages No. 1M and H17. In contrast, the tails of small phages No. 1M and H17 are different, since their protein composition in each phage pair depends on the tails of large phage particles which are antigenically different.

Our data suggest that, in both phages No. 1M and H17, the small phage genome is defective and codes for protein synthesis only for its own head and neck, whereas the tail is entirely borrowed from the respective helper phage. Thus, the small phage can be considered as a satellite of the large phage which produces com- plete particles of the small phage whose tail proteins are identical with those of the helper phage.

ADAMs, M. H. 1959. Bacteriophages. Interscience Pub- lishers, New York. p. 450.

SHORE, D., G. DEHO, J. TSIPIS, and R. GOLDSTEIN. 1978. Determination of capsid size by satellite bacteriophage P4. Proc. Natl. Acad. Sci. U.S.A. 75: 400-404.

TIKHONENKO, A. S. 1961. The fine structure of phage of Bacillus mycoides. Dokl. Akad. Nauk SSSR, 138: 1449-1451.

1970. Ultrastructure of bacterial viruses. Plenum Press, New York and London. pp. 147- 155.

TIKHONENKO, A. S., and I. A. BESPALOVA. 1961. On two forms of a phage of Bacillus mycoides. Mikrobiologiya, 30: 867 - 869.

YANAGIDA, M., E. BOY LE LA TOUR, A. STEINBERCER, and E. KELLENBERGER. 1970. Studies on the morphopoieses of the head of bacteriophage T-even. VIII. Multilayered poly- heads. J. Mol. Biol. 50: 35-58.

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