FEMS MicrobiologyLetters 83 (1991) 65-68
© 1991 Federation of European Microbiological Societies 0378-1097/91/$03.50
Published by Elsevier
ADONIS 037810979100413A
65
FEMSLE 04608
Biological characterization of the lytic cycle of actinophage bA7
in Streptomyces antibioticus
Luis A. D i a z , P i l a r G o r n e z , C a r l o s H a r d i s s o n a n d M . R . R o d i c i o
Departamento de Biologfa FuncionaL Area de Microbiolog{a, Uniuersidadde Ouiedo, Ouiedo, Spain
Received 23 June 1991
Accepted 27 June 1991
Key words: Actinophage ~bA7; Streptomyces antibioticus; Adsorption; One-step growth curve
1. S U M M A R Y
2. I N T R O D U C T I O N
Some basic parameters of the lytic development of phage 4~A7 in Streptomyces antibioticus
are described. One-step growth experiments
demonstrated that at 2 8 ° C ~bA7 has a latent
period of about 60 rain and an exponential growth
period of about 35 rain. The average burst size
ranged from 70-100 plaque forming units per
infected cell. At the same temperature 50% of
the virions were adsorbed to germ tubes of S.
antibioticus in about 10 rain. This corresponds to
an adsorption constant of 6.5 x 10 -~° m l / m i n .
The phage was unable to adsorb the host at other
stages of the life cycle (spores or mycelium).
Divalent cations are not required for ~bA7 stability but Ca 2 + proved to be essential for adsorption
and also for a later stage of the vegetative development of the phage.
Bacteria of the genus Streptomyces are common inhabitants of the soil. They have a complex
life cycle (involving sporulation, spore germination and mycelial growth) and are of commercial
interest because of their capacity to produce a
wide range of antibiotics. Actinophage (phage
that infect actinomycetes) are of increasing interest in the study of Streptomyces molecular biology
(see ref. 1, for a review). They are abundant in
nature and can be readily isolated from soil samples. From this source we have obtained several
actinophages which infect Streptomyces antibioticus, a producer of the macrolide antibiotic oleandomycin [2]. One of these phage, ~bA7, was characterized in some detail and data on its general
and molecular biology have been published [2,3].
The phage is temperate, showing a wide host
range. Like most Streptomyces phage it has a
polyhedral head (52 nm in diameter) and a long,
non-contractile tail (102 nm in length), ending in
a base plate. The phage genome is a 46.7-kb
double-stranded-DNA molecule with cohesive
ends. Two regions which are not required for
Correspondence to: M.R. Rodicio, Departarnento de Biologia
Funcional, Area de Microbiologia, Universidad de Oviedo,
33006-Oviedo, Spain.
66
plaque formation were located in the restriction
map: a 1.2-kb region at about 18 kb from the
conventional right-hand end of the linear-DNA
molecule, and a larger segment of about 6.2 kb,
close to the left-hand end. The D N A of the
phage can integrate into the chromosome of several Streptomyces strains to form stable lysogens.
Integration occurs through recombination between attachment sites on the phage (attP) and
host (attB) genomes. The qSA7 attP site has been
located near the left-hand end of the phage DNA,
close to the largest dispensable region.
In order to increase our knowledge of the
general biology of 05A7 and its interaction with
the host, we have established in the present work
the basic parameters of the lytic cycle as well as
the optimal conditions for the propagation and
preservation of the phage.
other stages of the developmental cycle of the
host, but spores were incubated for different times
(see below) before the addition of the phage.
3.3. One-step growth experiment
The experiment was carried out essentially as
described by Dowding [7]. Following a 20 rain
period of adsorption (see above) the infected
culture was filtered through 0.45-/xm filters (Millipore) and washed three times with 5 ml of
nutrient broth to eliminate free phage. Cells collected on the filter were resuspended by shaking
in 25 ml of nutrient broth with 100 mM Ca :+
(first infected culture, containing 10 7 cells/ml)
and an 0.5-ml sample was further diluted with 25
ml of the same medium (second infected culture,
with 2 × 105 cells/ml). Both cultures were incubated at 2 8 ° C with shaking and at 5-10 min
intervals samples were removed and immediately
assayed for plaque-forming units (pfu).
3. M A T E R I A L S A N D M E T H O D S
3.1. Bacteriophages, bacterial strains and general
techniques
&A7, originally described in [2], was the phage
used throughout this work and S. antibioticus
A T C C 11891 was the host for the propagation of
the phage. Methods for propagation, assay and
purification of the phage, and for culture of
Streptornyces were essentially as in [4], but GAlE
medium [5] was used for growth of S. antibioticus.
3.2. Adsorption
Adsorption rates of ~bA7 to young germ tubes
of S. antibioticus were determined by free-phage
titration [6]. Nutrient broth (Oxoid) containing
1% glucose was inoculated with 108 s p o r e s / m l ,
and incubated at 28 ° C with shaking, until short
germ-tubes were formed (about 6 h). Germinated
spores were mixed with phage at a multiplicity of
infection of 0.1 and then Ca(NO3) 2 was added to
a final concentration of 100 mM. The adsorption
mixture was kept at 28 ° C with shaking. Samples
were withdrawn every 5 or 10 rain, centrifuged at
12 000 r e v / m i n and free phage determined on S.
antibioticus top layers. The adsorption constant
(K) was calculated according to Adams [6]. The
s a m e method was used to assay a d s o r p t i o n at
4. R E S U L T S A N D D I S C U S S I O N
4.1. Plating and preserr, ation conditions
The effect of Ca 2+ and Mg 2+ on the efficiency
of plating (cop) of d,A7 was assayed by plaque
formation on lawns of S. antibioticus. Appropriate dilutions of the phage lysate were plated on
nutrient agar containing a range of MgC12 a n d / o r
Ca(NO3) 2 concentrations. The results obtained
are summarized in Table t. In the absence of
both Ca 2+ and Mg 2+, no plaques were detected
(cop < 1 0 - 4 ) . Ca 2+ was shown to be essential for
phage propagation. The size and number of
plaques increased with increasing concentrations
of this cation, with an optimum of 100 mM. This
seems unusually high, as the optimum for other
actinophages is in the range of 4-25 m M [4]. At
higher concentrations (data not shown) the outline of the plaques was difficult to determine
because the medium became turbid and very soft.
Mg :+ could not replace Ca 2+. Moreover, in combination with the optimal Ca 2+ concentration, a
range of Mg 2+ concentrations had no effect on
the plating efficiency of qSA7. However, at suboptimal concentrations of Ca 2+ addition of Mg :+
ted-to some improvernent in-~the cop. ..........................
67
Table 1
I n f l u e n c e o f d i v a l e n t c a t i o n s ( C a 2+ a n d M g 2 + ) o n O A 7
p r o p a g a t i o n ( S e v e r a l c o n c e n t r a t i o n s o f C a 2+ ( A ) a n d M g 2+
(B) w e r e t e s t e d in t h e a b s e n c e o f t h e o t h e r c a t i o n . 100 m M
C a 2 . w a s s e l e c t e d as t h e o p t i m a l c o n c e n t r a t i o n . F u r t h e r
i m p r o v e m e n t in t h e e o p w a s n o t a c h i e v e d by a d d i t i o n of
M g 2+ t o g e t h e r w i t h t h e o p t i m a l C a 2+ c o n c e n t r a t i o n ) .
A
j~P a~mc,
~""~Qe ~
eop b
B
MgSO 4 a
eop b
2
5
10
25
50
100
<10 4
<10 4
9 × 10 3
0.3
2.9
5.5
9.0
_
2
5
10
25
50
100
<10
<10
< 10
< 10
c
c
c
4
4
-4
4
" Final concentration (mM).
b T h e e o p w a s c a l c u l a t e d w i t h r e s p e c t to a c o n t r o l s u p p l e m e n t e d w i t h 8 m M C a ( N O 3 ) 2 a n d 10 m M M g S O 4.
c V e r y small p l a q u e s .
The influence of temperature on the eop of
~bA7 on S. antibioticus was also tested. The phage
produced plaques at 28 ° C, 34 ° C and 37 ° C, with
similar eop. However, the plaque size decreased
with increasing temperature, probably due to the
faster growth of the host. No plaques were formed
at 45 ° C (eop < 10 -6) although the host was able
to grow at this temperature.
Therefore, in accordance with these results,
100 mM Ca 2+ and 28 ° C were used in the experiments described below.
Since divalent cations are known to enhance
phage stability, the effect of Ca z+ on survival of
qSA7 was analyzed. Dilutions (10-~-10 5) of
phage in nutrient broth with and without added
Ca(NO3) z (100 mM) were stored at 4 ° C. At one
month intervals samples from each dilution were
removed and plated on S. antibioticus top layers.
After at least 6 months storage, titres did not fall
even in the absence of Ca 2+. Stability of OA7 in
the absence of divalent cations is in agreement
with the relatively high resistance of the phage to
treatment with chelating agents [3].
4.2. Adsorption
The adsorption of ~ A 7 to young germ tubes of
S. antibioticus was studied as described in MATERIALS AND METHODS, Plotting the percentage of
unadsorbed phage against time (Fig. 1) the curve
indicated that 50% of adsorption had already
occurred within about 10 min. The titre of free
phage continued to decrease for 50 rain and,
before 60 min it increased sharply, indicating lysis
of infected cells and release of newly synthesized
virions. Thus, the latent period established for
~bA7 by adsorption experiments is close to 1 h.
Ca 2+ proved to be essential for adsorption since
there was no loss of free phage during a period of
2 h in the absence of this cation.
Adsorption of OA7 at other stages of the life
cycle of S. antibioticus was also studied. Results
indicated that g,A7 particles only adsorb to germ
tubes of varying length (6-10 h of incubation at
28 ° C). Thus, the phage was unable to adsorb to
dormant, dark and swollen spores (0-5 h) or to
young and old mycelium (12, 24, 36 and 48 h). In
all these cases recovery of free phage was always
higher than 90%.
The fact that ~bA7 adsorption is limited to
germ tubes is noteworthy. Many actinophages,
including PK-66 [8], V P l l [7], Pal 6 [9] and the
well-characterized g, C31 [10,11], can also adsorb
to young mycelium. In this connection, it is relevant to point out that the limits between germ
tubes and mycelial growth are somehow artificial
(10-12 h of incubation at 2 8 ° C were arbitrarily
100
,.r
0.
)aJ
~o..o...o~ o ~
o ~ - - " " ~ ~ o
k
/
Qt
I
20
i
40
I
60
TIME
I
80
I
100
i
120
(rn I n )
Fig. 1. A d s o r p t i o n o f & A 7 to g e r m i n a t i n g s p o r e s o f S. antibioticus in the p r e s e n c e (e) a n d a b s e n c e (©) o f C a 2+.
68
set as the limit in this paper). Furthermore, little
is known about the changes which might occur at
the cell surface in the transition from one stage
to another. Therefore it would be interesting to
identify the host receptor for 4,A7, accessible
only in germinated spores. Whatever the nature
of this receptor, it does not seem to be available
in all Streptomyces species. For example, the
phage is unable to infect S. lil~idans due to adsorption failure (L.A. Diaz, unpublished results).
Adsorption rate constants of ~hA7 to germ
tubes of S. antibioticus were low, ranging between 1.0 × 10 -~ ml,/min (5 min after contact)
and 2.3 × 10-~° m l / m i n (50 min after contact).
This correlates well with the values reported for
most actinophage-actinomycete systems (around
10 - 9 m l / m i n ) [12], which are, in general, an
order of magnitude lower than those for coliphage [6]. It has been suggested that low rates of
adsorption for actinophages are due to asynchrony of the developmental cycle of the host and
the consequent variation in competence of the
population for phage infection [12]. This may also
be the case for ~bA7.
4.3. One-step growth curt.'e
The multiplication curve of actinophage qSA7
is shown in Fig. 2. It was determined by measuring the p f u / m l in serial samples of infected germ
100
/
10
I
I
I
I
20
40
60
80
TIME
I
100
I
120
(rain)
Fig. 2. One-step growth curve of phage ~A7 on S. antibioticus
ATCC 11891 at 28°C.
tubes of S. antibioticus. The curve gave a value of
60 min for the latent period, close to the one
obtained in adsorption experiments. The rise period was 35 rain, and the burst size varied between 70-100 pfu per infected cell. Release of
virions did not occur (less than 10 pfu per infected cell) when Ca 2+ was absent from the dilution medium (added to avoid secondary infections, see MATERIALS AND METHODS). C a 2+ therefore is not only required for adsorption but also
for a later stage of the intracellular cycle of &A7
in S. antibioticus.
ACKNOWLEDGEMENTS
L.A.D. was the recipient of a grant from the
Plan de Formaci6n det Personal Investigador of
the Ministerio de Educaci6n y Ciencia (Spain).
The work was supported by Grant PB-85-0403
from the C A I C Y T (Spain). The authors are
grateful to Dr. D.G. Swan and Dr. K.F. Chater
for helpful comments on the manuscript.
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158. Academic Press, London.
[2] Diaz, L.A., Hardisson, C. and Rodicio, M.R. (1989) J.
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[3] Diaz, L.A., Hardisson, C. and Rodicio, M.R. (1991) J.
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Bruton, C.J., Kieser, H.M., Lydiate, D.J., Smith, C.P.,
Ward, J.M. and Schrempf, H. (1985) Genetic Manipulation of Streptomyces - A laboratory manual. The John
lnnes Institute Foundation, Norwich, U.K.
[5] Hardisson, C., Manzanal, M.B., Salas, J.A. and Suarez,
J.E. (1978) J. Gen. Microbiol. 105, 203-214.
[6] Adams, M.H. (1959) Bacteriophages. Interscience, New
York.
[7] Dowding, J.E. (1973)J. Gen. Microbiol. 76, 163-176.
[8] Okanishi, M. and Okami, Y. (1966) J. Gen. Appl. Microbiol. 12, 207-217.
[9] Rosner, A. and Gutstein, R. (1981) Can. J. Microbiol. 27,
254-257.
[10] Lomovskaya, N.D., Mkrtumian, N.M., Gostimskaya, N.L.
and Danilenko, V.N. (1972) J. Virol. 9, 258-262.
[11] Suarez, J.E., Caso, J.L., Rodriguez, A. and Hardisson, C.
(1984) FEMS Microbiol. Lett. 22, 113-117.
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(1980) Microbiol. Rev. 44, 206-229.