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J. gen. Virol. (1984), 65, 2067-2072. Printed in Great Britain
Key words: B. subtilis phages/SPPl-related phages~phage homology~restriction patterns
Homology between Phages SPP1, 41c, 22a, p15 and SF6 of Bacillus subtilis
By M A R I O A. S A N T O S , 1'2 H E R M i N I A D E L E N C A S T R E 1'3.
AND L U I S J. A R C H E R 1'3
1Laborat6rio de Gen$tica Molecular, Instituto Gulbenkian de Ci~ncia, Oeiras, Portugal,
2Faculdade de Ci~cias de Lisboa and 3 Universidade Nova de Lisboa, Portugal
(Accepted 25 July 1984)
SUMMARY
Bacteriophages SPP 1, 4 lc, 22a, p 15 and SF6 of Bacillus subtilis share a common and
specific host receptor site for adsorption. Experiments described here have established
the relatedness between these phages. They were indistinguishable on the basis of hostrange, plating efficiency, various growth parameters and serological properties. In
addition, they shared the ability to carry out generalized transduction. They could be
differentiated, however, by the restriction patterns of their DNAs, with the exception
of 41c and 22a, which seemed to be identical. Recombination between 41c and SPP1
was demonstrated by transfection with mixed digests of their DNAs.
A mutation conferring resistance to infection by bacteriophage SPP1 (pha-2) was recently
mapped in the chromosome o f Bacillus subtilis 168 (Santos et al., 1983). Out often other bacteriophages tested, only 41c, 22a, pl 5 and SF6 failed to infect pha-2 strains. Although these phages
share some morphological characteristics with SPP 1, until now no evidence has been presented
as to their possible relatedness. In order to clarify the specificity of the pha-2 mutation, a comparative study of these five phages was undertaken. SP01, a phage unrelated to SPP1 (Hemphill
& Whiteley, 1975), was included as a control.
Bacterial strains used are listed in Table 1. Bacteriophages SPP1 (Riva et al., 1968), p15
(Jacobson & Landman, 1975), 41c (Zsigray et al., 1973), 22a (Jacobson & Landman, 1977), SF6
(Steensma & Blok, 1979) and SP01 (Okubo et al., 1964) were propagated on B. subtilis 168T + as
described previously (Santos et al., 1983).
Lysates were treated with 100 gg/ml DNase I at 37 °C for 30 min, before use in transduction
experiments.
M broth and M soft agar (Santos et al., 1983) were used for growth of bacteria, preparation of
phage lysates and plating.
SPP1, 41c, 22a, p15 and SF6 exhibit the same plaque morphology. They all form large, clear
plaques, sometimes surrounded by a turbid halo, when plated on an appropriate strain. The
Table 1. Bacterial strains
Strain
B. subtilis 168T+
Relevant properties
Prototroph
Use in this work
Propagation of phages
Plating bacteria
B. subtilis RUB808
lys-3 trpC2 metBlO gtaA Host-range
B. subtilis IGCgl06
lys-3 metBlO p h a - 2
Host-range
B. subtilis SLll
spoVB91 phe-12 trpC2
Recipientin transduction
B. subtilis SLI013
spolIA69 lys-3 trpC2
Recipientin transduction
B. subtilis W23
Prototroph
Host-range
B. subtilis 14593*
Prototroph
Host-range
B. amyloliquefaciens RUB501 EryR StrR RfmR
Host-range
B. licheniformis RUB503
EryRStrR RfmRL y s Host-range
B. pumilus RUB502
EryR StrRRfmRB i o Host-range
* Bacitracin-producing strain.
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0022-1317/84/0000-6130 $02.00 © 1984 SGM
Origin/reference
Yasbin et al. (1976)
Santos et al. (1983)
Lencastre & Piggot (1979)
Liu et al. (1982)
ATCC collection
Wilson & Young (1972)
Wilson & Young (1972)
Wilson & Young (1972)
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Table 2. Effect of deoxyribonuclease on the plating efficiency of phages SPP1, 41c, 22a, p15,
SF6 and SP01
Plating efficiency(~o)
DNase concn. ~g/ml)*
0
30
200
A
f
SPP1
100
64
28
41c
100
76
32
22a
100
52
31
"h
pl 5
100
58
33
SF6
100
77
46
SP01
100
106
104
* M soft agar (10 mi-CaCl2) supplemented with deoxyribonuclease.
host-range of these phages was tested by spotting different dilutions (103 to 101° p.f.u./ml) of
lysates on lawns of several Bacillus strains (see Table 1). From our survey, only B. subtilis 168T ÷
and its derivatives seem to be able to support phage growth. B. subtilis W23, B. subtilis
ATCC14593, B. licheniformis, B. amyloliquefaciens and B. pumilus were resistant to these phages
at all dilutions spotted. SP01, used as a control, showed a broader host-range, which included B.
subtilis W23 and B. subtilis ATCC14593. However, the e.o.p, on the former strain was only 10-4
when compared to B. subtilis 168T ÷. The phages under study share common host receptor sites
for adsorption (Santos et al., 1983). Accordingly, only SP01 could plate on a strain carrying the
pha-2 mutation, while SPP1, 41c, 22a, p15 and SF6 plated at normal efficiencies in a gtaA
mutant, resistant to SP01. Plating efficiencies depend not only on the particular host used but also
on the composition of the growth medium. Several steps in the growth cycle of phages 41c and
SF6 have been reported to be calcium-dependent (Landry & Zsigray, 1980; Steensma & Blok,
1979). In addition to this cation requirement, infection by 41 c was shown to be DNase-sensitive,
a feature not described for any other B. subtilis phage (Zsigray et al., 1973). We have therefore
analysed the influence of divalent cations and DNase on the e.o.p, of this group of phages. To
test for a DNase effect on plating efficiency, lysates were titrated on B. subtilis 168T ÷ using M
soft agar with 30 or 200 ~tg/ml DNase I. When the effect of divalent cations was investigated,
CaCI2, SrC12 or MgCI2 was added to non-supplemented M soft agar to a final concentration of
10mu. The infection of all phages but the control (SP01) had an absolute requirement for
divalent cations. Highest e.o.p, s were obtained with Ca 2÷ (100 ~) and, in decreasing order, with
Sr 2+ (70 to 80~) and Mg 2÷ (40 to 60~). The e.o.p, of all five phages was approximately the same
in each experimental condition. When DNase was added to the medium, a significant decrease
in the e.o.p, was obtained for all phages except SP01 (Table 2). DNase sensitivity cannot, however, be considered a feature unique to SPPl-related phages since we have found that SP02 and
4~105 are also DNase-sensitive (data not shown). Moreover, it has been reported that, following
adsorption, 2 D N A is rendered accessible to DNase during the first 6 min (Zgaga et al., 1973). It
is noteworthy that all DNase-sensitive phages seem to belong to Bradley's morphological group
B (phages with flexible, non-contractile tails), while phages not affected by this enzyme, namely
SP3, SP8, SP81, q~l, q~25, Tq~8A (Zsigray et al., 1973)and SP01 (our data), belong to group A
(phages with contractile sheets). It has been suggested that in 41 c the site of action of DNase is in
the area of contact between phage tail and cell wall surface (Zsigray et al., 1973). DNase sensitivity might thus be a common feature of group B phages whose D N A injection mechanisms
seem to differ from those of group A phages.
Latent period and burst-size values for SPP1, 41c, 22a, p15 and SF6 have been derived from
one-step growth experiments performed essentially as described by Adams (1959). Under our
conditions, the estimated latent period for these phages was 30 min and burst sizes ranged from
540 to 630 p.f.u./cell. These values are significantly higher than those previously reported. It has
been pointed out, however, that latent period and burst size depend more on the host strain and
environment than on the phage itself (Ackermann et al., 1978). In fact, we have observed that
the burst size of SPP1 under our conditions was 560, whereas it was 260 when TY medium
supplemented with 10 mM-MgC12 and 0.1 mM-MnC12 was used. This latter value is similar to
that reported by Klotz & Spatz (1971) who used the same medium.
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Table 3. General&ed transduction by phages SPP1, 41c, 22a, p15 and SF6*
Phage
Transduction
frequencyt
(Lys+)
Transduction
etficiency~
Co-transduction
(Lys+ Spo+) (~)
SPP1
5.3 × 10 -6
1.8 × 10 - 6
52.0
41c
22a
p15
SF6
1'2 x
7.5 ×
9.2 ×
1.2 x
2.6 x
2.3 x
3-2 x
4.1 x
52.5
52.8
68.9
56.0
10-5
10 -6
10-6
10-s
10 -6
10-6
10-6
10-6
* Strain SL1013 was grown in M broth to an absorbance (600 nm) of 0.8, centrifuged and resuspended in half the
original volume of the same medium. 0.1 ml of an appropriate dilution of the phage lysatewas then added to 1 ml of
culture. Input multiplicitiesranged from 1 to 2. Transduction mixtures were incubated for 15 min at 37 °C, centrifuged and resuspended in 1 ml minimal medium (Anagnostopoulos & Spizizen, 1961). Appropriate dilutions were
then plated on selective media. 400 Lys÷ transductants from each cross were replica-plated to Schaeffer plates
(Piggot, 1973) and incubated for 48 h at 42 °C to check the sporulation phenotype, Spo÷.
t Ratio of number of transductants to viable cells.
~:Ratio of number of transductants to phage particles.
Generalized transduction by SPP 1 has been repeatedly demonstrated (Ferrari et al., 1978;
Lencastre & Archer, 1980; Yasbin & Young, 1974). W e have found that lysates of 41c, 22a, p15
and SF6 share this capacity. Efficiencies and frequencies of transduction for Lys + were
determined, using strain SL1013 as recipient. The values obtained were similar for all phages
(Table 3). The co-transduction frequency between lys-3 and spoIIA69 was also checked since
this p a r a m e t e r is generally considered to be a measure of the size of transducing D N A (Henner
& Hoch, 1980). Co-transduction of these markers was consistently higher with p15 than with
other phages (Table 3). However, using strain SL11 as the recipient, we have found similar cotransduction frequencies between phe-A and spoVB for SPP1 and p l 5 (20 ~ average co-transfer
between these markers).
Conclusive proof of the phylogenetic relatedness of the phages studied here was obtained by
measuring their inactivation rate constants with anti-SPP1 serum (supplied by T. A. Trautner).
K values ranged from 430 min -1 (SF6) to 690 rain -1 (SPP1), thus establishing that the five
phages are antigenically related. Viability of SP01 particles was not affected by this serum even
in concentrations 103 times higher than those required for the inactivation of more than 9 0 ~ o f
SPP1 or its relatives.
In order to assess further the degree o f relatedness between these phages, D N A s were isolated
as described by Maniatis et al. (1982), digested with several restriction endonucleases and then
analysed by agarose gel electrophoresis. Although similar patterns were obtained, they were sufficiently different to allow a clear distinction between phages SPP1, 41c, p15 and SF6 (Fig. 1).
Phages 22a and 41c gave the same D N A restriction pattern with all enzymes tested so far (SalI,
KpnI, BglI, XbaI, SmaI and EcoRI) and should therefore be considered identical. The a p p a r e n t
molecular weight of the genomes, estimated from the sum o f their EcoRI fragments, was close to
42 kb for all phages (our unpublished data). A n interesting feature of these phages is the presence, in several digests, o f fragments in less than molar amounts and others appearing as diffuse
bands, containing molecules of heterogeneous size. In the case of SPP1, these features were
shown to be a consequence of the partial circular permutation and terminal redundancy of its
genome (Ratcliff et al., 1979).
Humphreys & Trautner (1981) have shown that transfection activity, absent in single
restriction digests of SPP 1 D N A , was recovered when competent cells were exposed to mixtures
of different restriction digests, provided that they contained widely overlapping fragments
which would recombine to form a complete genome. Accordingly, we assessed genomic
relatedness between our phages by carrying out transfection experiments with mixtures of a SalI
digest of SPP1 D N A and an XbaI digest of 41c D N A . SPP1 has two recognition sites for SalI
and one of the resulting fragments covers about 80 ~ of the genome (Ratcliff et al., 1979). XbaI
introduces only one cut in 41c D N A , yielding two fragments of similar size. Results from transDownloaded from www.microbiologyresearch.org by
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2
3
4
5
6
7
8
9
10
Fig. 1. Electrophoresis of Sail and BglI cleavage fragments of phage DNAs in a 0.8% agarose gel.
Lanes 1 to 5, Sail digests of DNA from phage SF6 (lane 1), p15 (lane 2), 41c (lane 3), 22a (lane 4) and
SPP1 (lane 5). Lanes 6 to 10, BglI digests of DNA from phage SF6 (lane 6), p15 (lane 7), 41c (lane 8), 22a
(lane 9) and SPP1 (lane 10).
T a b l e 4. Transfection activity o f restriction digests of wild-type SPP1 and 41c DNAs*
DNA
SPP1
41c
SPP1
41c
SPP1
41c
Restriction enzyme
None
None
Sail
XbaI
Sail ~
XbaI 5
Transfectants/ml
5-0 × 105
6-0 × 105
< 101
2.0 × 101
9.5 x 102
* Competent cultures of strain 168T ÷ were prepared according to Yasbin et al. (1973). Transfection mixtures
contained 1 p-g of DNA per ml of competent cells. In mixed digests, 0.5 ktg of DNA was used from each digest.
After shaking for 30 min at 37 °C, DNAase I was added to a final concentration of 100 ~tg/ml. After an additional
5 min appropriate dilutions were plated on M soft agar with 0.1 ml of B. subtilis 168T ÷.
fection e x p e r i m e n t s are s u m m a r i z e d in T a b l e 4. As expected, single restriction digests o f either
p h a g e h a d very little or no biological activity. H o w e v e r , a significant n u m b e r o f t r a n s f e c t a n t s
was o b t a i n e d w i t h m i x e d digests o f the two D N A s , i m p l y i n g t h a t r e c o m b i n a t i o n o c c u r r e d
b e t w e e n them. Fig. 2 shows the EcoRI restriction p a t t e r n o f two such r e c o m b i n a n t s c o m p a r e d
w i t h the EcoRI pattern of the p a r e n t a l types. A l t h o u g h s o m e differences exist b e t w e e n these rec o m b i n a n t s , m a i n l y in their larger fragments, they b o t h lack SPP1 f r a g m e n t s Eco-9 and Eco-12.
In addition, they b o t h h a v e in c o m m o n , w i t h 41c, a b a n d o f 2.2 kb (41c Eco-8 f r a g m e n t ) which,
a c c o r d i n g to our m a p p i n g results (unpublished), seems to be located n e a r the ' p a c ' site o f 41c
genome.
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2
3
4
Fig. 2. Electrophoresis of EcoRI cleavage fragments of D N A from phages SPP1, 41c and two
recombinants, in a 1% agarose gel. Lane 1, SPP1 D N A ; lanes 2 and 3, recombinant D N A s ; lane 4, 41c
DNA. Arrows indicate SPP1 fragments Eco-9 and Eco-12 (absent in both recombinants) and 41c fragment Eco-8 (present in both recombinants).
The experiments reported here clearly demonstrate that SPP1, 41c, 22a, p15 and SF6 are, in
fact, very closely related. The extensive homology between these phages makes them attractive
for studies on genome organization and evolution. Such studies will considerably benefit from
the existence of detailed physical and genetic maps for SPP 1 (Behrens et al., 1979; Ratcliffet al.,
1979) and physical maps for 41c, p15 and SF6 (our unpublished results).
We thank Dr H. Y. Steensma for the generous gift of phage SF6 and Dr O. E. Landman for the offer of phages
41c, 22a and p15. We thank Dr T. A. Trautner for many helpful discussions.
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