MiCrobiology (1 996), 142, 979-983
Printed in Great Britain
A bacteriophage of Rhodopseudomonas
blastica
H. Gorhamt and C. S. Dow
Author for correspondence: C. S. Dow. Tel: +44 1203 523539. Fax: +44 1203 523701.
e-mail: [email protected]
Department of Biological
Sciences, University of
Warwick, Coventry
CV4 7AS, UK
A bacteriophage, 4BHG1, was isolated from a small eutrophic pond from which
its host, Rhodopseudomonasblastica, was originally obtained. It is a lytic
bacteriophage specific for R. blastica which also causes non-specific lysis of
Rhodobacter sphaeroides 8253. 4BHGl has an icosahedral head of 62 nm
diameter and a short 39 nm tail. Caesium chloride density gradient
centrifugation of infected cell lysates gave a single bacteriophage band a t a
density of 1.385 g ~ m -but
~ , also occasionally a second band was observed a t a
lower density. No differences were apparent between bacteriophage taken
from either of the two bands. 4BHG1 contained double-stranded DNA with a
size of 48 kb and a G + C content of 50.6 mol%. The bacteriophage adsorbed to
both photosynthetically and chemoheterotrophically grown R. blastica a t an
identical rate of 1-39 x
ml-1mi+. One-step growth curves and kinetic
studies of the bacteriophage under these physiological regimes showed no
differences in the latent and rise periods and only slight changes in the burst
size. Adsorption of this bacteriophage is cell-surface specific and attachment
only occurs to the 'older' pole of the budding reproductive cell.
Keywords : Rhodopsetldomonasblastica, bacteriophage, polar adsorption, asymmetric
division, adsorption kinetics
INTRODUCTION
R hodobseudomonas blastica is a member of the
Rhodospirillaceae and was first isolated from a small
1
eutrophic pond (Eckersley & DOW,1980). Reproduction
is by a budding mode of growth followed by symmetrical
cell division. Cells are non-motile. R. blastica is capable of
growing either photosynthetically under anaerobic conditions or chemoheterotrophically under aerobic conditions. Few bacteriophage have been isolated for
members of the Rhodospirillaceae, the majority of which
have been specific for Rhodobacter sphaeroides. These
include bacteriophage RS1, a lytic phage isolated from
sewage (Abeliovich & Kaplan, 1974), 4RsG1 and R#l,
both temperate bacteriophage (Duchrow & G i f i o r n ,
1987; Mural & Friedman, 1974), and R46P (Tucker &
Pemberton, 1978), which exists as a plasmid during
lysogeny. A few bacteriophage have been isolated for
Rhodobacter capsulatus and their host ranges determined
(Schmidt e t al., 1974). RC1 is a virulent bacteriophage of
Rhodobacter capsulatus which has been used in one of the
t Present address: Nuff ield Department of Pathology and Bacteriology,
John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK.
0002-0190 0 1996 SGM
very few studies of the bioenergetics of bacteriophage
replication (Schmidt e t al., 1974). However, there is
relatively little information available concerning the
interaction of these bacteria with their respective
bacteriophage(s).
In this communication we report the characterization of
#BHGl, a lytic bacteriophage isolated from the same
environment as its host. We have investigated a number
of aspects of the interaction of 4BHG1 with its host,
including adsorption and replication under both chemoheterotrophic and photoheterotrophic growth.
METHODS
Organisms and media. The bacterial strains used were:
Rhodopsetldomonas blastica NCIB 11567 (Eckersley & Dow,
1980); Rhodobacter sphaeroidesNCIB 8253 ; Rhodobacter sphaeroides
subsp. cordata (Gest e t al., 1983); Rhodopseudomonas paltlstris
NCIB 8288 ; Rhodobacter capstllattls NCIB 8254; Escberichia coli
K12. R. blastica and other members of the Rhodospirillaceae
were grown routinely in a pyruvate-malate salts medium
(Whittenbury & Dow, 1977) supplemented with 0.1 % (w/v)
yeast extract (PMY). Batch cultures were incubated at 30 "C in
a gyratory shaker. During photoheterotrophic growth, light
was provided by tungsten lamps at an intensity of 40 p E m-' s-'.
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979
H. G O R H A M a n d C. S. D O W
Bacteriophage isolation and purification. 4BHG1 was isolated
from a small eutrophic pond by the water filtration method of
Logan e t al. (1981). After enrichment, 4BHG1 was isolated from
a crude lysate of R. blastica and purified after infecting a midexponential-phase culture to an m.0.i. of 0.01. After 48 h
incubation, chloroform was added to the culture and cell debris
was removed by centrifugation at 3000 g . Caesium chloride was
added to a mean density of 1.546 g cmP3 and the lysates were
centrifuged at 240500g (Beckman SW40 rotor) for 36 h at
10 OC. Opalescent bacteriophage bands were removed with a
syringe and needle by insertion through the side of the tube.
Samples were dialysed against PBS (g I-': NaC1, 8.0; KC1, 0.2;
Na,HPO,, 1-15; I<H,PO,, 0.2; pH 7 at 4 "C). 4BHG1 was
stored at 4 "C over 100 pl chloroform (ml sample)-'.
Preparation of bacteriophage antiserum. Phage 4BHG1
(0.5 ml 10'' p.f.u. ml-' in PBS), caesium chloride purified, was
emulsified with an equal volume of complete Freund's adjuvant
and injected into rabbits. After a period of 6 weeks the rabbits
were bled and antiserum was prepared from the resulting serum.
Adsorption rate and one-step growth experiments. The
adsorption rate of 4BHG1 to R. blastica was determined by the
method of Adams (1959). The protocol for the one-step growth
curves was as follows. 4BHG1 was added to chemoheterotrophically or photosynthetically grown R. blastica to an rr1.o.i.
of 0.02. The bacteriophage/cell suspension was incubated
without shaking at 30 "C for 50 min. Crude antiserum (raised
against 4BHG1) was added to a 50-fold dilution of this
suspension. After a further 5 min incubation, the cell suspension
and
in prewarmed medium and
was diluted to lo-,,
samples were removed at various time intervals for titration of
4BHG1.
Titration of 4BHG1. Serial dilutions of bacteriophage suspension were added to PMY top agar (PMY with 0.7 %, w/v,
Difco agar) containing 0.2 ml of an early-stationary-phase
chemoheterotrophically grown R. blastica culture. Plates were
routinely incubated at 30 "C chemoheterotrophically. This was
justified as there was no perceived difference in titration data
between photoheterotrophic and chemoheterotrophic incubation.
Bacteriophage DNA isolation, size determination and base
composition. D N A was isolated and purified by the method of
Kirby et al. (1967) with an additional final 2-propanol precipitation step. After desiccation, the D N A was resuspended in
a minimal volume of TE buffer (10 mM Tris, p H 8.0; 1 mM
EDTA). The mol% G + C content of q5BHG1 D N A was
determined by the buoyant density method of Mandel e t al.
(1968) using a Beckman Model E analytical ultracentrifuge. E .
coli K12 D N A (density of 1.720 g ~ m - ~was
) included as an
internal standard.
The size of 4BHG1 D N A was determined after endonuclease
digestions as described by Maniatis etal. (1982). D N A fragments
were separated on 0.8% (w/v) agarose gels.
Electron microscopy. After negative staining with 1'30 (VJ/V)
phosphotungstic acid, pH 7, samples were examined using a
J EOL 100s transmission electron microscope.
RESULTS AND DISCUSSION
Bacteriophage isolation
Purification of 4BHG1 by caesium chloride density
gradient centrifugation gave a single opalescent band.
A260and
measurements (using a Philips PU 8720
UV/VIS spectrophotometer) and titration of gradient
fractions against the host bacterium showed this
980
1 -40
X
U
t
.-
1.38
Q,
.-
t;
2 1.36
rc
e
1 *34
Fraction no. (2 ml)
Fig. I . Buoyant density determination of 4BHG1 by caesium
chloride centrifugation. Caesium chloride was added to the
bacteriophage preparation to give a mean density of
1.546 g ml-'. Centrifugation was a t 240500 g a t 10 "C. The
peak in bacteriophage concentration (2 x lo9 p.f.u. ml-')
corresponded with those for A
,,,
and A
.,,
0,
Refractive index;
0,
A,,,;
A,,,.
.,
to correspond to bacteriophage with a density of
1.385 g cm-3 (Fig. 1). Occasionally, however, two bacteriophage bands were observed of 1.385 g cm-3 and
1-384g ~ m - Both
~ . bands contained viable bacteriophage,
and electron microscope studies and protein and DNA
analyses failed to distinguish between these bands. Similar
observations have been made with bacteriophage P1
when isolated by caesium chloride density gradient
centrifugation (Sternberg e t al., 1977), with the upper
band being, as in this study, approximately 90% of the
total bacteriophage population. No physical differences
between the bacteriophage of the two bands could be
determined ; consequently, both bands were pooled.
Plaque formation and host range
Using standard overlay methods, 4BHG1 formed plaques
of varying size on R. blastica, ranging from 0-5 to 1.5 mm
in diameter with a slight halo. Bacteriophage isolated
from these large and small plaques were found to give rise
to varying plaque sizes when replated. The e.o.p., defined
as the number of plaques under one set of conditions
divided by the number of plaques under a standard set of
conditions, was independent of the growth physiology of
R. blastica, i.e. chemoheterotrophic or photoheterotrophic ; consequently, 4BHG1 was routinely titred on
chemoheterotrophically grown cells.
Seeley & Primrose (1980) noted that coliphage did not
show coincidence of the temperature for maximum e.0.p.
of the bacteriophage and the maximum specific growth
rate of the host. They showed that coliphage fell into
three physiological classes depending upon the effect of
temperature on their e.0.p. These three classes, low
temperature, mid-temperature and high temperature,
appeared to be stable properties of the virus and were not
influenced by the temperature for growth of the host.
However, bacteriophage 4BHG1 appeared to differ in
that the temperature for optimal e.0.p. was directly related
to the temperature for the maximum specific growth rate
of the host, i.e. 30 O C .
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Rhodopsezjdomonas blastica bacteriophage
Fig, 2. Electron micrograph of negatively stained (1 %
phosphotungstic acid) 4BHG1. Bar, 100 nm.
Of the phototrophic species investigated, 4BHG1 was
found to replicate only in R. blastica; however, nonspecific lysis, i.e. lysis due to attachment of bacteriophage
to the cell surface rather than intracellular replication of
the bacteriophage, occurred with Rhodobacter sphaeroides
8253.
Characteristic of other Rhodospirillaceae bacteriophage so
far isolated, 4BHG1 showed a very limited host range.
Characterization of 4BHGl DNA
The nucleic acid within the capsid of 4BHG1 was
identified as double-stranded DNA, as determined by
restriction endonuclease digestion. Linearized 4BHG1
DNA gave rise to a discrete band on agarose gel
electrophoresis, with a size of 48 kb. The buoyant density
of the bacteriophage D N A was calculated to be
1.724 g ~ m - ~corresponding
,
to a G + C molar ratio of
50.6 Yo. This value varies significantly from that of its
host, R. blastica, which is 65-3mol YO(Eckersley & DOW,
1980). Such differences are not unusual within
bacteriophage-host systems of the Rbodospirillaceae. For
example, Rhodobacter sphaeroides 2.4.1 has a G C content
of 67 mol% while two of its bacteriophage, 4RSG1 and
4RS1, have values of 46 and 71.8 mol Yo, respectively
(Abeliovich & Kaplan, 1974; Duchrow & Giflhorn,
+
1987).
Bacteriophage morphology
4BHG1 has a polyhedral head of 62 nm diameter and a
short tail of 39 nm (Fig. 2). N o tail fibres were seen and
the tail appeared to be non-contractile. The morphological
features of 4BHG1 place it in Group C of Bradley’s
classification scheme (Bradley, 1967). The polyhedral
head and short tail resemble those of bacteriophage RC1,
which infects Rhodobacter capszllatzls. These two bacteriophage differ considerably from other bacteriophage isolated for Rhodobacter sphaeroides, RS1, R41, 4RsG1, R46P
and R49 (Abeliovich & Kaplan, 1974; Duchow e t al.,
Figrn
3. Attachment of 4BHG1 t o R. blastica during the cell cycle.
Samples were stained with 1 % phosphotungstic acid. Bar,
500nm. a, Bacteriophage; E, cell envelope growth point; X,
cell division site.
1985; Mural & Friedman, 1974; Pemberton & Tucker,
1977; Tucker & Pemberton, 1978), which characteristically possess long tails up to 116 nm in length.
Bacteriophage attachment
Electron micrographs (Fig. 3) clearly show that 4BHG1
attaches to one specific pole of the R. blastica cell, the ‘ old ’
pole of the budding cell (Eckersley & DOW,1980).
Bacteriophage can be seen attached to both poles when
the cell approaches symmetrical division, i.e. when both
poles can be designated ‘old’. The bacteriophage did not
adsorb to the actively growing pole nor to the plane of
division. The relationship between bacteriophage adsorption and the R. blastica cell cycle is shown in Fig. 3.
There would, therefore, appear to be macromolecular
differentiation of the cell surface of R. blastica by which
4BHG1 attaches only to the ‘old’ pole. Only one other
bacteriophage for the Rhodospirillaceae has been reported
to have site-specific adsorption, Rpl, which is specific for
Rhodopsezldamonaspalzlstrisle5. This bacteriophage attaches
only to the new or dividing pole (Bosecker e t al., 1972).
4BHG1 and Rpl are therefore markers of macromolecular
differentiation of the cell surface of their respective hosts.
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98 1
H. G O R H A M a n d C. S. D O W
n
J
A
I
1
10
0
20
L
L
30
L, , - - l
40
50
k
\
l
60
70
Time (min)
.. .............. .... ... .................. ....... .............. ...............,,....... .............. ....... ............. .............. ...... .... .... .....
Fig. 4. Rate of adsorption of 4BHG1 (lo7p.f.u. m1-l) t o cells
(5x lo8 m1-l) of chemoheterotrophically (0)
and photohet:erotrophically (0)
grown R. blastica at 30°C. The results of a
representative experiment are shown.
5l
50
100
150
200
250
Time (min)
Fig, 5. Representative one-step growth curves of 4BHGl under
varying growth regimes. Exponential phase cultures of R.
blastica were diluted t o a cell concentration of 5 x lo8cells rnl-l.
Bacteriophage were added and allowed to adsorb (m.0.i. of
0.02 for 50 min a t 30 "C) before the addition of bacteriophage
antisera (1: 50, 5 min a t 30 "C) and dilution with prewarmed
medium. Samples were subsequently assayed for p.f.u. Under
chemoheterotrophic conditions (+), infected cells vvere
maintained in the dark aerobically; under photoheterotrophic
conditions (0)the infected cells were illuminated
(40 pE mw2s-') and maintained under anaerobic conditions.
Electron micrographs of 4BHG1 attached to Rhodobmter
sphaeroides 8253 showed that the bacteriophage adsorbed
non-specifically to the cell surface.
Adsorption kinetics and one-step growth curves
Adsorption rates for 4BHG1 appeared to be identical for
both chemohetero trophically and photosynthetically
grown cells (Fig. 4). The adsorption constant k .was
determined as 1-39x
ml-' min-' with a R. blaxtica
population density of 5 x 10' cells ml-l. Above this cell
density, the rate of adsorption was constant. Unlike
q5BHG1, 4RS1 exhibits some difference in the rate: of
adsorption between photoheterotrophically and chemo982
The growth regimes had little o r no effect upon the length
of the latent and rise periods and little effect upon the
burst size (Fig. 5). The burst sizes under chemoheterotrophic and phototrophic growth were 25 & 2.5 and
30 2.1, respectively. The latent period was between 80
and 100 min and the rise period was 100 min, irrespective
of the growth regime. The effect of the mode of growth
on the burst size has been studied in only two other
Rhodospirillaceae phage, RC1 and RS1 (Schmidt et al.,
1974; Abeliovich & Kaplan, 1974), and as with 4BHG1
the burst size is unaltered. However, the former do
display differences in the latent and rise periods which are
not evident in R. blastica.
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2
'0
heterotrophically grown cells. Photoheterotrophic cells
appeared, to a large extent, to be immune to infection.
This partial immunity may represent changes in the
structure or composition of the surface of the cells, these
changes affecting the adsorption or penetration process of
the bacteriophage. Further investigations showed that
adsorption was independent of the cations Mg2+, Ca2+,
NH; and Na+. Only three bacteriophage of the
Rhodospirillaceae, 4RC1 (Schmidt e t al., 1974), 4RsG1
(Duchow et al., 1985) and 4RS1 (Abeliovich & Kaplan,
1974), have been reported to require Ca2+ and Mg2+.
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environments and possible changes in the mode of growth
physiology of its host, R. blastica.
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Received 7 July 1995; revised 5 October 1995; accepted 23 October 1995.
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