J. gen. Virol. (I976), 32, I29-I32
I29
Printed in Great Britain
Characteristics of Phage AP50, an R N A Phage
Containing Phospholipids
(Accepted IO March 1976)
SUMMARY
A bacteriophage specific for Bacillus anthracis was isolated and designated as
AP5o. The nucleic acid of phage AP5o is RNA and the virion contains five different
phospholipids. Some physical and biological characteristics of the phage, including
morphology, were examined. To the best of our knowledge, this RNA bacteriophage containing phospholipids is the first to be isolated for a Gram-positive host.
Franklin (I974) has published a list of bacteriophages which contain or are presumed to
contain lipids. Olsen, Siak & Gray (I974) and Bradley & Rutherford (I975) recently described some lipid-containing phages which lyse strains of pseudomonads and Enterobacteriaceae carrying R plasmids.
A bacteriophage designated as AP5o was isolated from the soil by one of us (G.I.) with
the aid of streptomycin technique (Ivfinovics & Lantos, I958), by using as host a vaccine
strain Sterne proved to harbour a defective prophage (Nagy & Iv~,novics, 1972). Phage AP5o
appeared to be a phage with some unusual characteristics (Nagy, I974). We therefore
undertook a detailed study of this phage and confirmed its extreme host-specificity; only
one third of 34 strains of Bacillus anthracis were lysed by the phage. None of 52 strains
belonging to 6 different Bacillus spp. were sensitive to AP5o.
When propogated in a liquid culture of Bacillus anthracis strain Sterne (CN 18-74 cur) in a
yeast extract-peptone medium (Csisz/tr & Ivfinovics, I965), phage AP5o reached a p.f.u.
value of ~ IOl°/ml. To concentrate the phage material, the centrifuged lysate was passed
through a Millipore filter (pore size o'45 #m); 5 ~ (w/v) polyethylene glycol (PEG) and
o'5 M-NaC1 were then added to the filtered lysate (Yamamoto et al. I97O). Since the virion of
AP5o appeared very sensitive (Nagy, I974), the procedures were carried out with special
care. The fine precipitate formed within a few hours on addition of PEG was centrifuged
5ooo g for zo min, and the pellet was resuspended in a buffer (Rabussay, Zilling & Herrlich,
~97o) the volume of which was I ~ of that of the lysate. This phage material was subjected
to DNase and subsequently RNase treatment (each 5/~g/ml). Further purification was
achieved by cyclic ultracentrifugation. Finally, the phage material was layered onto a preformed sucrose gradient (2o to 6o ~, w/v) and centrifuged in a Janetzki VAC 6o ultracentrifuge (SW 5 rotor) for 18o min at 8oooo g. Fractions were assayed for infectivity and
their n~~ values were measured with a Zeiss-Abbe refractometer. The peak fractions were
pooled (p.f.u. : IoU/ml) and dialysed against either buffer or distilled water for electron
microscopy and chemical analysis respectively. Since hypertonic CsC1 degraded the virions
within 24 h, a pre-formed CsC1 density gradient (2o to 8o ~, w/v) was used to measure the
buoyant density of the phage AP5o. Centrifugation was carried out as described above.
Table I lists the main physical and chemical characteristics of phage AP5o. Shaking for
3o min with either chloroform (5 ~, w/v) or ether (25 ~ , w/v) inactivated the phage to a
survival of about I x lO-4. The lipid extract of phage AP5o was analysed on a silica gel G
thin-layer plate in a two-dimensional system, according to Abramson & Blecher 0964). The
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I3o
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Fig. i. (a) Intact AP50 phage particles from the peak fraction of a sucrose density gradient,
negatively stained with 2 % (w/v) uranyl acetate. (b) Purified particles exposed to CsC1 for 24 h and
negatively stained with 2 ~ (w/v) uranyl acetate.
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Short communications
~31
Table 1. Physical and chemical properties o f phage AP5o virions
Buoyant density in sucrose
Buoyant density in CsCI
RNA content
DNA content
Protein content
Lipid content
l "198 g/ml
1"3o7 g/ml
65 #g/ml (Schneider, I957)
Nil (Burton, 1956)
683 #g/ml (Lowry et aL 195I)
I4"3 % of the dry weight (Bligh & Dyer, 1959)
lipid extract gave five different spots, four of which could be identified as phosphatidyletharlolamine, phosphatidic acid, cardiolipin and phosphatidylglycerol. One spot could not be
identified. At 37 °C the latent period was 55 min and the average burst size was 31 o p.f.u./cell.
The adsorption rate constant to strain Sterne was measured at t'5 +_o'5 × xo-9 ml/min.
In preliminary electron microscopical studies by Nagy (I974) with concentrated suspensions of phage AP5o, the virions showed a considerable variation in their morphology. We
assumed that the different appearances of the individual particles may have been caused
by degradation occurring during the manipulations involved in electron microscopy. That
this is the case was proved by making the electron microscopical preparations with the phage
material taken from the peak fraction of the sucrose gradient. Fig. 1(a) shows a very homogeneous, electron-dense hexagonal virion population, the particles being 8o nm in diam. The
virions of AP5o were obviously easily degraded with different non-specific agents. The most
striking effect could be demonstrated by exposing the virions to hypertonic CsCI solutions
for several hours. When the phage material purified by sucrose gradient was mixed with
CsCI at a final concentration of 5o ~ and kept for 241a at 4 °C, the infectivity was virtually
lost, parallel with a profound morphological change of the virions. Approximately half of
the particles appeared to have a tail-like structure and all virions had partially or completely
lost their nucleic acid. The capsid of the degraded virions exhibited a double-layered structure
from which emerged a tail-like structure or a hole, presumably caused by the removal of
this structural element (Fig. I b). Evidence indicates, however, that phage AP5 o does not
have a tail; what appears to be a tail is really a distorted virion with an artifact resembling
a tail-like structure. This may result from effects which lead to an extrusion of nucleic acid
and the elastic lipoprotein capsid, possibly at a site corresponding to the receptor which
dilates and temporarily accommodates a proportion of the nucleic acid. We are inclined to
believe that phages PR3 and PR4 (Bradley & Rutherford, I975) also do not have a noncontactile tail, but rather an artifact resembling a tail.
We should like to thank Mrs Judith Nagy and Miss Judith Szendi-Horv~th for skitled
technical assistance.
Institute of Microbiology
Medical University
Szeged, Hungary
EL1SABETHNAGY
B. PR~,GAI
G. IV,~NOVICS
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(Received 3o October I 9 7 5 )
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