The Adsorption of Phage to Group N Streptococci. The Specificity of

J. gen. ViroL 0968), 3, Io3-H9
I03
Printed in Great Britain
The Adsorption of
Phage to Group N Streptococci. The Specificity of Adsorption
and the Location of Phage Receptor Substances
in Cell-wall and Plasma-membrane Fractions
By J. D. O R A M * AND B. R E I T E R
National Institute for Research in Dairying, University of Reading
(Accepted I6 February I968)
SUMMARY
The specificities of adsorption and multiplication of phages attacking
some strains of lactic streptococci (serological group N) were investigated.
At multiplicities of infection (m.o.i.) of o. I to I p.f.u./coccus, the specificity of
adsorption was similar to that of multiplication, but some strains also adsorbed one or more h¢terotogous phages. At m.o.i. ~ IOO both homologous
and heterologous strains of streptococci were lysed from without. The
specificity of adsorption to cell walls was the same as to whole cocci and,
with the exception of 2 phages, was irreversible at 3o°. Three types of phage
receptors, with different specificities, were recognized in the cell wall. Extraction of streptococci with lipid-solvents did not affect the adsorption of those
phages which were irreversibly adsorbed to cell walls but reduced the adsorption of other phages. The plasma membrane of Streptococcus lactis strain ML 3,
but not the cell wall, inactivated phage ml 3 but not heterologous phages in
the presence of electrolytes. The plasma membrane of a phage-resistant
mutant of this strain did not inactivate phage although it was similar in
chemical composition to that of the parent strain.
INTRODUCTION
The concept of specific phage receptors in bacterial cell surfaces is now generally
established, largely as a result of the pioneering studies of Burnet (I934), Jesaitis &
Goebel (1952) and Weidel, Koch & Lohss (I954). However, specific phage receptor
substances have as yet been demonstrated only in the cell-envelopes of the enterococci
in the form of complex lipoprotein and lipopolysaccharides. Although phage-inactivating substances have been obtained from some Gram-positive bacteria, including a
group C streptococcus (Krause, I958 ), Staphylococcus aureus (Rosato & Cameron,
1964) and Streptococcus faecium (Vidaver & Brock, I966), the specificities of the
phage-inactivating processes have not been examined in detail.
Many of the phages attacking the lactic streptococci possess fairly restricted host
ranges (Nichols & Hoyle, 1949; Whitehead & Bush, 1957) and adsorb well to the host
cells. The effects of physical factors, e.g. electrolyte concentration, pH value and
temperature on the adsorption of a Streptococcus lactis phage were established by
Cherry & Watson (I949), and we have shown (Reiter & Oram, 1962) that suramin
inhibits the adsorption of phage to S. lactis. However, the specificity of adsorption of
phage to lactic streptococci and the nature of the phage receptor substances have not
been previously investigated.
* Present address: The Microbiological Research Establishment, Porton, Wiltshire.
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J.D. tRAM
A N D B. R E I T E R
This paper deals with the specificity of phage adsorption and the location of specific
phage receptor substances within the surface layers of lactic streptococci. The isolation and chemical characterization of the different types of phage receptor substances
will be reported later.
METHODS
Bacteria. The following strains of Group N streptococci were obtained from the
National Collection of Dairy Organisms (catalogue numbers are given in parentheses):
Streptococcus cremoris strains np (6o7), KH (I 198), TR (I200), E 8 (I I96), 699 (699),
799 (799), 803 (803), D 9Streptococcus lactis strains ML 3 (763), C I t (509), 712 (7IZ), M2SI (701).
They were grown in glucose-Lemco broth (GL broth) or in Difco trypticase-soya
broth (TS broth) at 3°o as described by t r a m & Reiter (I965).
Phages. Phages for the streptococci (indicated as ml 3, c zo, 7z2, etc.) were also
obtained from the National Collection of Dairy Organisms. Stock suspensions of
phage were prepared by making serial dilutions of the dried phage preparation in
I t ml. of autoclaved litmus milk and inoculating these with o.r ml. of a I t -~ dilution
of a 24 hr culture in milk of the host streptococcus. The infected cultures were incubated at 30° from 6 to I8 hr and those showing lysis were pooled, acidified with
sterile N-lactic acid, centrifuged at 3000 rev./min, for 30 min. and the supernatant
neutralized with sterile N-NaOH. After centrifuging, the supernatant was stored at 4 °.
Phage counts were made as previously described ( t r a m & Reiter, I965). The stock
suspensions contained between I × m 8 and 5 × Ioa° p.f.u./ml.
Preparation of cell walls. Cocci grown in GL or TS broth at 3°o were harvested in the
late exponential phase of growth and washed twice with and resuspended in k strength
Ringer's solution in o.oi M-potassium phosphate buffer, pH 6.8 (Ringer-phosphate
solution). The cocci were broken by shaking with glass beads, either for 30 min. at o °
in a centrifuged shaker head (Shockman, Kolb & Toennies, I957) or for 4 min. in the
Braun disintegrator (Shandon Ltd, London), cooled by intermittent jets of COs. The
broken suspension was diluted with 4 vol. of ice-cold Ringer-phosphate solution and
filtered through a sintered glass funnel to remove the glass beads. The glass beads were
washed with cold Ringer-phosphate solution and the washings added to the filtrate
and the combined suspension of broken cocci centrifuged at ~o,ooo rev./min. (the
M.S.E. rotor no. 24o7 was used throughout in a Superspeed 4o centrifuge at 5 °) for
3o rain. The deposit was washed twice with cold M-KC1, resuspended in o.I M-potassium phosphate buffer (pH 6.8) and then centrifuged several times at 2ooo rev./min.
for Io min. to deposit unbroken cocci. The cell walls were then washed once with
o.I M-potassium phosphate buffer (pH 6.8), suspended in o'o5 M-tris+HC1 buffer,
(pH 8.o) containing 5o/~g./ml. of deoxyribonuclease and incubated at 37° for 3 hr in
the presence of chloroform. The cell walls were then washed once with o'o5 M-tris+ HC1
buffer (pH 8-o), resuspended in the same buffer containing 5o/~g./ml. of trypsin and
incubated at 37° for I8 hr, in the presence of chloroform. They were finally washed
three times with deionized water and freeze-dried.
Preparation of plasma membranes. The supernatant obtained by centrifuging the
disintegrated cocci at io,ooo rev./min, for 3o min. was centrifuged at 21,ooo rev.]
min. for 2 hr. The deposit, which contained membranes, cell wall fragments and much
nucleic acid, was washed once with o.2 M-KC1in O'oI M-tris + HC1 buffer (pH 7"5) and
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Adsorption of phage to group N streptococci
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resuspended in 50 ml. of a 20 % (w/v) solution of sucrose in o.oI M-tris+HC1 buffer
(pH 7"5) and I2"5 ml. portions were layered on to 65 ml. of a 35 % (w/v) solution of
sucrose in the same buffer in Ioo ml. polypropylene centrifuge tubes. The tubes were
centrifuged at zI,ooo rev./min, for z hr. and the material which layered at the density
boundary was carefully removed with a syringe, without disturbing the deposit. The
deposit, which contained mostly cell wall but some membrane material, was discarded. The low-density material was dialysed against 3 1. of o.ooI M-tris + HC1 buffer
(pH 7"5) at 4 ° for I8 hr and deposited by centrifuging at zI,ooo rev./min, for 2 hr. After
two more cycles of this procedure (centrifuging in sucrose solutions, followed by
dialysis and re-centrifugation), a homogeneous, translucent and slightly orangecoloured preparation of membranes was obtained from which characteristic cell walt
constituents, e.g. rhamnose, ghicosamine and muramic acid, were absent. The membrane preparations contained 3 % to 6 % of ribonucleic acid and although most of this
could be removed by further washing with deionized water, extensive washing appeared
to change the properties of the membrane, which became opaque in appearance and
sedimented in much smaller centrifugal fields (io,ooo rev./min, for I hr). As similar
changes occurred on freeze-drying, membrane preparations were normally suspended
in deionized water and stored at - 2 o °.
Extraction of streptococci with lipid solvents. Ceils from 5o ml. of a I6 hr culture in
GL broth were washed successively with IOO ml. of Ringer-phosphate solution and
IOO ml. of deionized water and resuspended in z.o ml. of deionized water. One half of
the suspension (the 'control') was diluted with Ringer-phosphate solution to an
I ella.
E~80mu
value of o'4 and stored at 4 ° and the other portion was extracted by one or
other of the following procedures.
Alcohol and ether. The suspension (I.O ml.) was cooled to - 2 o ° and mixed with
15 ml. of absolute alcohol at - 2o% After 3o min. the mixture was placed in a water
bath at 37° for 60 min. After centrifuging, the cocci were resuspended in 25 ml. of
peroxide-free diethyl ether and incubated at 37° for a further 6o min. After a second
centrifugation, the cocci were suspended in the same volume of Ringer-phosphate
solution as the 'control' and ether removed by bubbling with N~.
n-ButanoL The cell suspension was diluted to 5"o ml. with deionized water and
shaken at room temperature with 5 ml. of n-butanol saturated with water for 3o min.
The extracted cells were deposited by centrifugation and washed successively with
to ml. each of n-butanol saturated with water, ethyl alcohol and ether and then
resuspended in Ringer-phosphate solution as described above.
Gelfiltration. A 9o x 2"5 cm. column of Sephadex G-zoo was prepared and used at 4 °
as described by Andrews (t965). For the determination of molecular weights, a
standard curve was prepared by plotting the logarithms of published molecular
weights of crystalline proteins against their elution volumes, measured either by their
enzymic activities or extinctions at 225 m/z. The following proteins were used to calibrate the column (molecular weights x lO-3 are given in parentheses), fl-Galactosidase
from Escherichia coli (51 o to 53o), bovine 3' globulin (I 50 to 17o), alkaline phosphatase
from E. coli (75 to 8o), ovalbumin (44 to 46) and cytochrome c from horse heart
(I2"4).
Thin-layer chromatography of lipids. Samples of lipid dissolved in benzene were
applied to thin layers (o'5 mm.) of silica gel HF 254 and 366 (E. Merck Ag. Darmstadt,
German Federal Republic) spread on 2o x 20 x o'3 cm. glass plates and previously
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J . D . O R A M A N D B. R E I T E R
activated by heating at lO5° for I hr. The plates were chromatographed for 30 rain.
using benzene + diethyl ether + ethylacetate + acetic acid (80: Io: lO: o-2, v/v) as the
solvent in the narrow solvent chamber system of Storry & Tuckley (1967). The plates
were dried and sprayed with a solution of 0"005 % rhodamine B and 0.025 % dichloro-R-fluorescein in methanol and examined under ultraviolet light (254 and
366 m#).
Paper chromatography of sugars. Portions (5 to IO rag.) of the plasma membranes of
streptococci were hydrolysed with I.O ml. of 0. 5 N-HC1 in an atmosphere of N2 at
IOO° for I8 hr and dried in vacuo over P~O5 + NaOH. The residue was dissolved in
25 to 50/*1. of deionized water and 5/,1. portions spotted on to Whatman No. I paper
and chromatographed in n-butanol + pyridine + acetic acid + water (60:4o: 3: 20, v/v)
or ethyl acetate + pyridine + water (2: I : I, v/v, top phase). Carbohydrates were located
by the reagent of Trevelyan, Proctor & Harrison (I95O).
Adsorption of phage. (a) By viable streptococci. Cultures of streptococci in GL broth
were harvested after incubation at 3°o for 18 hr, washed once in ¼ strength Ringer's
solution and resuspended in o.I M-NaCI + o.ooi M-CaClz+ o.oI % (w/v) gelatin to give
1,0 e m
an Egs0muvalue of 0"4, which corresponded to about 3.1o8 cocci/ml. The suspension of
cocci (2.0 ml.) and a suspension of phage (about IOs p.f.u./ml.) in the same medium
were separately equilibrated at 30° for IO rain. and I-O ml. of phage suspension added
to the cocci. The adsorption mixture was shaken gently for 15 rain., centrifuged at
about 8,000 g for 3 rain. and dilutions of the supernatant in ¼ strength Ringer's solution counted in duplicate to determine the number of unadsorbed p.f.u.
(b) By cell walls. This procedure was similar to that employed for viable cocci except
that, as the number of unadsorbed p.f.u, was, with the exceptions reported in the
' Results' section, unaffected by centrifuging the adsorption mixture, the centrifugation
step was omitted.
(c) By plasma membranes. In this procedure the volume of the adsorption mixture
was reduced in order to conserve material. After temperature equilibration, o.1 ml. of
phage suspension (about lO7 p.f.u.) was added to 0"4 ml. of membrane suspension and
the mixture incubated at 30° for 30 min. The titre of the residual phage was determined
without centrifugation. In preliminary experiments more consistent results were obtained when the NaCl+CaCl2+gelatin medium was replaced by GL broth and
although later found to offer no advantage, the latter medium was employed, except
where stated in the text. A unit of phage receptor activity was defined as the amount of
material which adsorbed or inactivated lOs p.f.u, under these conditions and the
specific activity as units of phage receptor activity/mg.
Chemical analyses. Before estimating the protein and RNA contents of membrane
preparations, the membranes were suspended (4 mg./ml.) in deionized water and dispersed by ultrasonic disintegration (Salton & Freer, 1965). Protein was estimated by
the method of Itzhaki & Gill (I964), except that the mixtures of membranes and reagents were centrifuged at about 5000 g for IO rain. to deposit insoluble material before
the extinctions were measured. RNA was measured, after hydrolysis with 5 % (w/v)
trichloracetic acid at 9°o for 20 rain., by the orcinol reaction (Mejbaum, 1939). The
lipid contents of membrane preparations were determined by extracting 20 mg. of
membrane, dispersed in I.O ml. of deionized water, with 25 ml. of chloroform+
methanol (2: I, v/v) at 60° for 20 min. The extract was filtered through a sintered glass
filter, washed with 0.2 vol. of 0"04% MgCI2 (Folch, Leese & Sloan-Stanley, I957),
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A d s o r p t i o n o f p h a g e to g r o u p N s t r e p t o c o c c i
evaporated to dryness in a stream o f Nz and then dissolved in chloroform + methanol
(2: I, v/v) and transferred to tared weighing bottles. After drying with Nz and then in a
desiccator over P20~, the weight o f the extracted lipid was estimated gravimetrically.
After hydrolysis o f samples with 4 N-HC1 at IOO° for I hr, glucosamine and m u r a m i c
acid were separated o n c h a r c o a l + c e l i t e columns (Perkins, I965) and determined by
the m e t h o d o f Strominger, Park & T h o m p s o n (I959). R h a m n o s e was determined by
the m e t h o d o f Gibbons (I955). After hydrolysis o f samples with 0"5 N-HC1 at Ioo ° for
I8 hr, glucose was determined with glucose oxidase (Hugget & Nixon, I957) and
galactose with the ' g a l a c t o s t a t ' reagent (Worthington Biochemical Corporation,
Freehold, N.J., U.S.A.).
RESULTS
Comparison o f the specificities o f adsorption and multiplication o f phage
Initially, we sought to establish the importance o f phage adsorption patterns on the
specificity o f phage multiplication. The latter was established by examining the ability
o f a phage to p r o d u c e plaques rather than to lyse cultures o f streptococci since some
o f these were lysed-from-without by high concentrations o f phage or phage-associated
muralytic enzymes (Oram & Reiter, I965). The ability o f a streptococcal strain to
support the multiplication o f the various phages was examined by plating the streptoTable I. Phage relationships o f some lactic streptococci; the
specificities o f phage multiplication
Strain of
streptococcus used
in phage assay
S. cremoris 699
S. cremoris ~
S. cremoris TR
S. cremoris 799
S. cremoris D 9
S. cremoris we
S. lactis
~ 3
Titration values (log. p.f.u./ml.) of phages using different
strains of streptococcus as host
,
^
,
699
kh
tr
799
d9
hp
ml 3
9"1o
8"7I
o
o
o
o
o
8"78
8'71
o
o
o
o
o
0
O
9"48
O
5"65
0
0
o
o
o
9'45
o
6"5z
o
O
O
943
O
8"57
0
0
o
o
o
o
o
8"o5
o
o
o
o
o
o
o
9"34
Table 2. Adsorption o f phage to streptococci
Residual p.f.u, as percentage of 'control' titre.
Phages
Strain of
streptococcus
S. cremoris 699
S. cremoris KH
S. cremoris TR
S. cremoris 799
S. cremoris D 9
S. cremoris He
S. lactis
ML 3
^
699
kh
tr
799
I I9"5
47"5] 98"o
92"4
14"8 _ 0"9-[ lO2"2 IOI'9
80"5 m9"3 ] z3"o
I5"5
104"5
96"7
..98:5......I 5 "I
9"4
1.7o:8.........69:51
97"0
d9
hp
ml 3
87"5 1o7"5 lO2"5
lOO"2 97"I
84"5
I9"8 56"8i......6"8:5"i
I6' 5 [
84"0
I02"I
[ I6"2 I lO5"7 ]4~-[
88"7
102"0
102" 5
102"5
90"O
~ 3'0
38"8J
97"9
lO3"5
95"I
96"8
lO4-1
t 3"1
3"7 I
Adsorption mixture containing about 3 x io s coccci and 2 to 13 x io 7p.f.u, in 3"o ml. of adsorption
medium were shaken at 3o° for I5 rain., centrifuged, and the supematant fluids titrated for residual
p.f.u. Cocci were omitted from the 'controls'. Residua of phages adsorbed are shown in boxes. Results
are averages of duplicate experiments.
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I08
J.D. tRAM
A N D B.
REITER
COCCUSwith dilutions of the different phages. Each phage had a fairly restricted host
range, none multiplying in more than two of the streptococcal strains (Table I). However, a lower specificity was found for the adsorption of phage, most phages being
adsorbed by several strains of streptococcus (Table 2). Thus, Streptococcus cremoris TR
adsorbed phage 799 in addition to its homologous phages tr and d 9 and S. cremoris 799
also adsorbed the heterologous phages tr and d 9. Similarly, S. lactis ML 3 adsorbed the
heterologous phage hp. Some homologous strains of phage were, however, adsorbed
with a much lower efficiency under these conditions. Thus, although the titre of phage
tr was the same when plated on strain TR or d 9, only about 3o ~ of the phage was
adsorbed by the latter strain. The low efficiency of plating of phage hp on strain o 9
appeared to be due to the very poor adsorption of the phage to this strain. Some strains
of lactic streptococci, other than those included in Tables I and 2, appeared to adsorb
a wide variety of heterologous phages but this was an extremely variable phenomenon
which was possibly the result of a non-specific inactivation of phage and not phage
adsorption.
Lysis-from-without of streptococci by heterologous phage
It was previously shown that Streptococcus lactis ML 3 was lysed-from-without by
high multiplicities of phage ml 3 ( t r a m & Reiter, 1965) and it was of interest to
determine the effect of this phage on heterologous strains of streptocci. Some strains
of lactic streptocci were found to be even more susceptible to lysis-from-without than
S. lactis ML 3 (Fig. I). The relative susceptibilities of the strains to lysis-from-without
by ml 3 phage were very similar to those previously found for their lysis by ml 3 phage
lysin or the lytic enzyme associated with the phage particles ( t r a m & Reiter, 1965).
Specificity o f # a g e adsorption to cell walls
Cell walls of several strains of lactic streptococci adsorbed phage irreversibly at 3o°.
The specificity of adsorption to the cell walls was, with some exceptions, similar to that
observed with washed streptococci (Table 3). The main exceptions were phages ml 3
(Fig. 2) and d 9, neither of which appeared to adsorb to the cell wall of the host strain
if the adsorption mixtures were not centrifuged before plating. Hence, the adsorption
of these phages to the host cell wall appeared to be reversible at 3o°. However, the cell
wall of Streptococcus lactis ML 3 adsorbed the heterologous phage hp and that of S.
cremoris D 9 adsorbed the homologous phage tr and the heterologous phage 799.
Weidel (1958) proposed that the adsorption of coliphage T I, which is unable to adsorb
irreversibly to the isolated cell envelope of Escherichia coli B, involves a mechanism
which functions in the living cell but not in the isolated cell envelope. However, before
digestion with trypsin or nucleases, the crude cell wall preparations of S. lactis ML 3
adsorbed phage ml 3 irreversibly, so this possibility was excluded. It seems more
likely that the receptor substances for phages ml 3 and d 9 were destroyed or removed
during the isolation and purification of the ceil walls.
Isolation of ml 3 phage receptor substances
The phage receptor activity of crude cell wall preparations of S. lactis ML 3 was not
affected by digestion with RNase or DNase but appeared to be destroyed by digestion
with trypsin. Subsequently, however, it was found that the supernatant fluid of the
trypsin-digested cell wall inactivated phage ml 3 irreversibly at 3o°. No loss in phage
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Adsorption o f phage to g r o u p N s t r e p t o c o c c i
1o~
-
I09
I
k
E
i
10 6
~o
l"Ok~
~
~
~
~.3
o
-
°.,
0.2F
I
i
I
•
I~
o
10,*
30
I
4o
o.4
•
Time (min.)
Amount of cell wall (mg.)
Fig. I
Fig. 2
Fig. I. Lysis-from-without of strains of group N'streptococci by phage ml 3. One ml. of a
suspension of phage in o'I M-potassium phosphate buffer (pH 6.8) was added to 4"o ml. of
suspensions of cocci in the same buffer at 37 °. Multiplicity of infection = IOO p.f.u./coceus;
E ~ ; ~ =extinction at 580 mF/cm:. C), S. cremoris 803 ; 0, S. lactis, c lO; D, S. lactis, ML 3.
Fig. 2. Adsorption of phage by cell walls of some lactic streptococci. Mixtures of cell wall and
phage in 3"oml. of o.I i - N a C l + o . o i i-CaCl~+o'oI ~o gelatin were shaken at 3o ° for
15 min. Mixtures were not centrifuged before diluting and plating of residual p.Lu. The
cell wall+phage mixtures were ©, S. cremoris I ~ + hp ; 0, S. cremoris KI-l+ kh ; D, S. lactis
ML 3+m13.
T a b l e 3. Adsorption o f p h a g e to streptococcal walls
Residual p.f.u, as percentage of 'control' titre
Phages
Strain of
streptococcus
S.
S.
S.
S.
S.
S.
S.
cremoris
cremoris
eremoris
eremoris
cremoris
eremosis
laetis
699
KH
TR
799
o 9
r
'
699
kh
] 4"5
I 3"9
87"0
lO4"4
109"6
o[
rip
118"5
ML 3
Io3"4
Oj
109"I
76"4
I03"O
92"9
I20,8
tr
799
•
d9
82"7 IO3"8
90"9
81"2
96"3 II1"6
[ 27"3
32"2 ] 101"5
]3o-1
4I.O[
80"8
[ O'I
32"3 I 82"3
93"4
lO7"9
93"9
1 2 6 " 5 1 0 3 " 9 100.8
hp
ml 3
lO7-8
85"2
87"O
82-0
97"6
~
Io..~
I I I-O
106"1
104"2
86"I
90"9
93"5
89"5
Adsorption mixtures containing I.O mg. of cell wall and 3 to I I x I C p.f.u, in 3"o ml. of adsorption
medium were shaken a t 3 o ° for I5 min. and titrated for residual p.f.u. Cell walls were omitted from
' controls'. Phages adsorbed by each type of cell wall are shown in boxes and these are average values
from two experiments; other results refer to single experiments.
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J . D . ORAM AND B. REITER
titre occurred when phage was incubated with trypsin alone at a concentration o f
IOO #g./ml. A 1 % suspension of the crude cell wall in o'05 M-tris + HC1 buffer (pH 8.o)
containing 5o Fg./ml. of trypsin was incubated at 37 ° for 18 hr; after centrifuging, the
supernatant fluid was dialysed against 2 changes of deionized water (IOO vols) at 4 °
and the non-diffusible material freeze-dried. When IOO rag. of this material was
filtered through a column of Sephadex G-2oo equilibrated with o.I M-KCI, the phageinactivating activity was obtained in the first of two protein-containing peaks (Fig. 3).
Comparison of the elution volume of the phage-inactivating material with those of
proteins of known molecular weight indicated that it had a molecular weight in the
range 2oo,ooo to 4oo,ooo. As this material contained approximately equal amounts o f
lipid and protein it seems probable that it was a lipoprotein derived either from the
cell wall or the plasma membrane.
I
I
I
I
i~
Blue dextran
n
I
2"10 ~'
!
t
I
th/-Galactosidase
%
3
.o
100
..=, 7.Globul
e~
.o
°~
~
,0 " ,
/
/
1
t
/
(~ Phosphatase C~
-
\_
k~
-
",'V
O
-
"*, y<o h,o2o
o
~
0
,
100
200
I \it,
300
ill ° ~ _ 1 o ,
400
500
Volume of eluate (nil.)
Fig. 3. Gel-ffitration of the supernatant obtained by digesting the cell wail-plasma membrane
fraction of S. lactis ML3 with trypsin. Ioo rag. of the supernatant was filtered through a
9o x 2"5 era. column of Sephadex G-2oo equilibrated with o-i u-KCI and etuted with the
same solvent at a flow rate of 2o ml./hr, at 4°. ©, E~&~,; @, phage inactivation; n, column
calibration (because of its carbohydrate content, y globulin behaves as a protein with a mol.
wt of about 21o,ooo (Andrews 1965)).
Crude ceU wall preparations contained between 6 % and IO ~/o lipid and therefore
appeared to contain a considerable amount of either cell wall lipoprotein or membrane
material. Hence, strain ML 3 appeared to possess two types of phage receptors--one for
phage hp located in the cell wall and another for phage ml 3, of uncertain location, but
containing lipoprotein. Moreover, as the activity of the lipoprotein-containing phage
ml 3 receptor was not destroyed by trypsin, it seemed possible that lipoproteins were
also present in phage receptors in the cells walls of some strains but that these were not
released by trypsin.
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Adsorption o f phage to group N streptococci
III
Effect of lipid solvents on the adsorption o f phage by washed streptococci
E x t r a c t i n g cells w i t h l i p i d s o l v e n t s s h o u l d d e c r e a s e t h e a c t i v i t y o f p h a g e r e c e p t o r s
w h i c h c o n t a i n e d l i p o p r o t e i n a n d w e t h e r e f o r e e x a m i n e d t h e effect o f n - b u t a n o l o r
a l c o h o l a n d e t h e r o n t h e a d s o r p t i o n o f p h a g e b y several strains o f s t r e p t o c o c c i ( T a b l e 4).
T a b l e 4, Effect of extracting streptococci with lipid-solvents on phage adsorption
Streptococcus
S.
S.
S.
S.
S.
S.
S.
S.
lactis ME 3
lactis M2 SI
lactis 712
cremoris we
cremoris 799
cremoris D 9
cremoris TR
cremoris TR
log. p.f.u./ml.
~-~
~
•
Residual phage
Input
Control Extracted
phage
cells
cells
Solvent* Phage
A
A
ml 3
m2 si
A
712
A
A
hp
799
d9
B
d9
B
tr
A
7"53
7"63
8"08
6"63
7"27
6"72
7"5I
7"35
5"87
6"47
7"29
5"14
6"61
6"44
7"04
6"58
Adsorption (%)
---~-
,
Control
cells
Extracted
cells
97q
92"5
82'5
97"0
78"5
47"6
66'5
83"5
26-o
52"I
81.2
97"3
63"5
7"6
o
78"0
7"40
7"32
7"35
5"1o
6"83
6"69
7"53
6"70
Ratio
extracted/
control
cells
0-27
0'56
0"98
I"O4
o'82
o'16
0
0"94
* Washed streptococci were extracted with (A) ethyl alcohol followed by diethyI ether or (B) nbutanol, as described in the text.
Adsorption mixtures containing approximately 3 x io s cocci/ml, and phage (as shown) were
shaken in 3'0 ml. of GL broth at 30 ° for 15 min.
T a b l e 5. Isolation o f phage ml 3 receptor substance f r o m
broken cells o f S. lactis ML 3
Cell fractionation procedure
Broken cells centrifuged IO,OOOrev./
min. x 30 min.
Supernatant (cytoplasm, plasma membrane and cell wall fragments)
Centrifuged 21,000 rev./min, x 2 hr
Supernatant fluid (cytoplasm)
Deposit (membrane and cell wall
fragments)
Centrifuged 21,000 rev./min, x 2 hr ir
35 ~ sucrose:
membrane from 1st cycle
membrane from 2nd cycle
membrane from 3rd cycle
Dialysed and centrifuged 21,000 rev./
min. x 2 hr; combined supernatants
Combined washings of 3rd membrane
fraction
Purified membranes
Dry
weight
Volume (rag./
(ml.)
ml.)
142o
--
I37O
52
-20"3
58
58
55
2340
I3'7"
lO.2"
8"4*
--
275
--
20
22"5
Phage
receptor
activity
(units/
ml. x lO-3)
Total
phage
receptor
activity
(units x
io -a)
4"29
6090
0"33
iio.o
481
5720
5"42
95"0
42"5
18"o
0"22"~
5500
2470
990
515
9"oi
4"05
1"8o
--
o'161
44
39"1
782
Specific
activity
(units/
rag. x io -s)
1"74
* Calculated from dialysed membranes.
t Average values.
Cells harvested from 40 1. of a tryptone-soya broth culture were washed twice with Ringer-phosphate
solution and disintegrated in the Braun cell homogenizer and the plasma membrane purified as
described in ' Methods'.
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II2
J.D.
ORAM
AND
B. R E I T E R
This reduced the extent of adsorption of phage m13 to S. lactis ML 3 from 97 % to 26 %
but had no effect on the adsorption of phage hp. Similarly, treatment with n-butanol
completely eliminated the adsorption of the homologous phage d 9 to S. cremoris
strains D 9 and TR but had little effect on the adsorption of phage tr. Extraction of three
others strains, S. lactis 712, S. cremoris I ~ and S. cremoris 799 had no significant effect
on their ability to adsorb their homologous phages but reduced the adsorption of
phage me si to S. lactis M2st by 44%. These results indicate that phage d 9 receptor
substance (and possibly that of phage me s like that of phage ml 3) contained lipid
but that the receptors of phages, hp, 799 and 7xe did not.
I
I
I
I
I
I
I
.I"1
El-
10 6
o
10 s
"N
01
0
0,2
I
0.4
I
0.6
I
0.8
I
1.0
I
1.2
Amount of material (mg.)
Fig. 4. Inactivationof phageml 3 by cellfractionsof S. lactis ML3. Phagewas incubatedwith
he plasma membrane (©), cell wall (O) or cytoplasmicfraction (D) in 0"5 ml. of GL broth
at 30° for 30 min.
Location o f phage ml 3 receptors
Streptococcus lactis ML3 was disintegrated in the Braun cell homogenizer and cell wall
and plasma membrane fractions purified as described i n ' Methods'. Although the cell
wall did not adsorb phage ml 3 and the cytoplasmic fraction (obtained by centrifuging
the broken cocci at 2I,OOOrev./min, for 2 hr) possessed very low phage-inactivating
activity, the phage was inactivated by very small amounts of the plasma membrane
(Fig. 4). One #g. of the most active preparation of purified membranes inactivated
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Adsorption o f phage to group N streptococci
113
about I0 ~ p.f.u., but before purification the specific activity of the membrane preparations were approximately 5 times higher than that of the purified material (Table 5).
The considerable loss in phage-receptor activity during purification of the membrane
was much greater than the amount of activity released into solution during the purification procedure (Table 5). As activity was also lost on freeze-drying, which also caused
changes in the physical properties of the membranes (see 'Methods'), membrane suspensions were normally stored at -2o%
Specificity of phage inactivation by the plasma membrane of Streptococcus lactis ML 3
The phage receptor activity of the plasma membrane of strain ME 3 was found to be
extremely specific. Thus, out of seven phages tested, only phage ml 3 was inactivated
by the membrane (Fig. 5). Neither phage 7zz, which was homologous for strain ME 3
Table 6. Chemical composition of the plasma membranes of
S. laetis ML 3 and ML 3/5 and S. cremoris 799
S. lactis ML 3
Protein
Lipid
RNA
Glucose
Galactose
Rhamnose
Glucosamine
M u r a m i c acid
48"z
36"0
2"9
0"9
0.6
o
o
o
S. lactis ME 3/5 S. cremoris 799
50"8
42"9
5"8
0"4
0'4
o
Trace
o
55"6
30"0
4"7
N.D.
r~.D.
o
o
o
N.D. : present b u t n o t determined quantitatively
and adsorbed by its cell wall, nor phage d 9, which appeared to adsorb to a membrane
type of receptor, was inactivated. The phage receptor activity of phage ml 3 resistant
strains of S. lactis ME 3 was also examined. Although about 9o % of the resistant
mutants obtained from a phage ml 3 lysate of strain ME 3 adsorbed the phage, two
mutant strains (ME 3/5 and ML 3/15) did not. The membranes isolated from these
mutants possessed very little or no phage receptor activity (Fig. 6) indicating that phage
receptors were absent from or non-functional in the membranes of these resistant
mutants. Strains ME 3/5 and ML 3/I5 were not resistant to phage 7ze, however, and
adsorbed this phage to the same extent as the parent strain, thus demonstrating the
independence of the genetic control of the phage ml 3 and 7zz phage receptor
substances.
The chemical composition of the membranes of the phage resistant strain ME 3/5 was
similar to that of the phage-sensitive parent strain, ME 3 (Table 6). Both contained
rather more lipid (about 40 %) than reported for the membranes of other species of
Gram-positive bacteria (Kodicek, 1962; Salton & Freer, I965), about 50 ~o protein and
from 3 to 6 % RNA. The plasma membrane of S. cremoris 799 contained less lipid
(30 %) than those of S. lactis ML 3 and ML 3/5. The lipids of the membranes of these
strains were examined by thin-layer chromatography. They contained large amounts
of phospholipids and fatty acids, small amounts of trigylcerides but little or no monoor di-glycerides, sterols or sterol esters. Hence the types of lipids in the membranes of
lactic streptococci appear to be similar to those reported for other bacterial membranes (Weibull, 1957; Kodicek, 1962).
8
J. Virol. 3
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J . D . O R A M A N D B. R E I T E R
ll4
10~,
r;
a
° '°iI"
•-~
10 s
I
I
I
I
t
100
200
300
400
500
Amount of membrane (Fg.)
Fig. 5. Specificity of the phage inactivating activity of the plasma membrane of S. lactis rcm 3.
The phages were incubated with 25 to 500 Fg. of the membrane in 0"5 ml. of GL broth at 30°
for 30 min. Phages: O, ml 3; e , kh; [3, ms 5 r; II, hp; A, 7zz; A , e 8; V , d 9.
I
I
I
Q
I
I
10
I
20
I.
30
Io' i
0
.=
10 ~ -
10 ~
0
Time ( ~ . )
Fig. 6. Absenc~ of phage receptor activity in plasma membranes of phage-resistant mutants
of S. lactis ML 3. O, Membrane of phage-sensitive strain ML 3, I00/~g.; @, membrane of
phage-resistant mutant ML 3/5, 500 #g.; [~, membrane of phage-resistant mutant, ML 3/I5,
500 #g.
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Adsorption o f phage to group N streptococci
II 5
Cation requirements for ml 3 phage receptor activity
Like other phages, ml 3 does not adsorb to washed cocci in the absence of mono- or
divalent cations and like other streptococcal phages (Reiter, I949; Shew, 1949) requires divalent cations for multiplication but not for adsorption. We therefore examined the effect o f Na + and Ca ~+ ions on the reversible inactivation of phage ml 3 by
the plasma membrane of S. lactis ML 3- The phage-inactivating activity of the membrane
was approximately the same in o.I ~i-NaCl+o.oI % gelatin as in G L broth but no
inactivation occurred in deionized water (Fig. 7). The addition of o.oz M-CaCI~ did not
affect the amount of phage inactivated by the membrane, indicating that Ca ~+ was
not required for this stage of the infective process.
I
I
I
I
0 106
lO ~
0
!
I
2
I
4
I
6
n.
8
Amount of plasma membrane (/zg.)
Fig. 7. Effect of cations On inactivation of phage ml 3 by the plasma membrane of S. lactis
ML3. Mixtures of phage and membrane in 0"5 mL of O, GL broth; A, deionized water;
D, o.I M-NaCl+o-oi .%0gelatin, or Q, o'I M-NaCI+o'oI M-CaCl~+o.oi % gelatin, were
incubated at 30° for 30 min.
DISCUSSION
At multiplicities of infection between o.I and I-O p.f.u./coccus the adsorption of
phage to lactic streptocci was found to be a fairly specific process. Nevertheless, most
strains of coccus adsorbed one or more heterologous strains of phage. This situation is
not confined to the lactic streptococci, many strains of staphylococcus, for example,
adsorb hcterologous strains of phage (Morse, I965). The inability of an irreversibly
adsorbed phage to multiply may be due to the failure o f the phage to complete later
stages in its maturation cycle or, in the case.of a temperate phage, because of lysogenic
immunity. However, we have been unable to show that phage hp is temperate for
S. lactis ML 3, in spite of its irreversible adsorption to that strain, and we know of no
8-2
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I I6
J. D. ORAM AND B. REITER
case in which lysogeny has been rigorously demonstrated in group N streptococci.
Both homologous and heterologous phages adsorbed to the cell walls of some strains.
The observed specificity of the adsorption of phages to streptococci is consistent with
the existence of several types of phage receptor, each having a characteristic specificity.
Thus phages tr and 799 were adsorbed by the cell walls of S. cremoris strains XR,
799 and o 9, indicating that both attach to a common receptor, present in all three
strains. Phages 699, kh and k were all adsorbed by cell walls of S. cremoris strains 699,
KH and K which, therefore, appeared to possess another type of receptor. Similarly, cell
wails of S. lactis MY3 and S. cremoris ~_Pwere found to possess a receptor specific for
phage hp. Doubtless many other types of phage receptor exist in the cell walls of other
lactic streptococci and although we did not find more than one type of receptor in
any one cell wall, only a limited number of strains were examined and others might
well possess several types. We have recently purified phage-receptor substances from
the cell walls of some lactic streptococci and their properties will be described in a
later paper.
Phage ml 3 attached only reversibly to the cell wall but was irreversibly inactivated by
the plasma membrane of S. lactis ME 3. As only phage ml 3 was inactivated, the membrane also determined the specificity of adsorption to the host cell. Several strains,
e.g. ME 3, D 9 and TR, appeared to contain both cell-wall and plasma-membrane
types of phage receptor. This appears to be the first report of the location of phage
receptor material in a bacterial plasma membrane although there is some evidence that
a similar situation exists in the closely related group D streptococci. Hence, phage was
found by Timperley, H o r n e & Stewart-Tull (1966) to adsorb to heat-killed cells but
not to the cell wall of Streptococcus faecalis and the phage-inactivating particles obtained by the action of lysozyme on Streptococcusfaecium by Vidaver & Brock (I966)
contained both cell wall and membrane material. As the phage-receptor activity of the
particles from S. faecium was sensitive to heat and treatment with mild acids or
alkali, to 6 M-urea and proteolyfic enzymes (differing in this last respect from the S.
lactis ME 3 phage receptor substance), Vidaver & Brock 0966) concluded that protein
was an integral part of the phage receptor. Phage P 3 was adsorbed by cell wall preparations which Vidaver & Brock (1966) obtained by the differential centrifugation of
mechanically disintegrated cells of S. faecium. However, we have found that although
it was relatively easy to obtain membranes of group N streptococci free of cell wall
components by differential centrifugation in sucrose solutions, it was very much more
difficult to obtain cell walls free of membrane material. Even after three cycles of differential centrifugation followed by extensive washing with buffers and water, 6 out
of 7 preparations of the cell wall of S. lactis ME 3 were heavily contaminated with
membrane material, the removal of which required either digestion with proteinases or
extraction with detergent. Our (unpublished) studies on the action of muramidases on
the cell wall + plasma membrane fraction of disintegrated cells of S. lactis ME 3 indicate the presence of some type of attachment between the ceil wall and the plasma
membrane, which may explain why spheroplasts rather than protoplasts are produced
when group N streptococci are digested with muramidases (Smith & Shattock, 1964).
Our initial failure to appreciate the intimate association between the cell wall and
plasma membrane of strain ME 3 resulted in misdirected attempts to characterise a
cell-wall-protein phage receptor and delayed considerably the location of the receptor
in the plasma membrane. A similar type of association between the cell wall and plasma
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Adsorption o f phage to group N streptococci
117
membrane of S. faecium could account for the occurrence of cell wall material in the
phage receptor particles obtained from spheroplasts by Vivader & Brock (1966).
The activity of the phage ml 3 receptor was markedly reduced by extracting cells
of S. lactis ML 3 with lipid solvents and this also destroyed the phage d 9 receptors of
S. cremoris D 9, suggesting that this phage receptor also occurred in the plasma
membrane. However, the destruction of a phage receptor by lipid-solvents is in itself
no proof that the receptor occurs in the plasma membrane, as the receptors for coliphages T2 and T6 are cell wall lipoproteins (Weidel et al. 1954) and those ofT3, T4 and T7
are cell wall lipopolysaccharides (Jesaitis & Goebel, 1952). Hence, the inability of
alcohol- and ether-extracted cells of Corynebacterium diphtheriae to adsorb phage B
(Kerszman, 1966) may have been due to an effect of lipid-containing substances in the
cell wall rather than in the plasma membrane.
The phage-inactivating activity of membranes of Streptococcus lactis ML 3 appears
to be considerably higher than that reported by Morse (1965) for the cell wall of
Staphylococcus aureus. Morse (1965) found that I #g. of staphylococcal wall inactivated about lO4 p.f.u, whereas in our experiments I/,g. of the membrane of S. lactis
ML 3 inactivated about lO6 p.f.u, and the specific activity of unpurified membrane
preparations was approximately 5 times greater. As the efficiency of plating of phage
ml 3 is only about 0-o5 to o. IO (tram, 1965) the number of particles inactivated by the
membrane was probably even greater than our results indicate.
We have considered the possibility that the rnl 3 phage receptor was not an integral
part of the plasma membrane but became loosely associated with, or adsorbed to, the
membrane during the disintegration of the cocci. However, this would not explain the
loss of phage-receptor activity during the purification of the membranes, as these
losses greatly exceeded the amount of activity released into solution during the purification procedure. It therefore seems probable that the observed loss of activity was due
to the destruction of some labile component of the phage receptor. Recently a lipoprotein, containing only traces of carbohydrate and no nucleic acid constituents, was
obtained from the membrane of strain ML 3 and possessed the phage receptor material
of this organism (tram, unpublished results).
It is generally accepted, by analogy with the coliphages, that adsorption of phage is
followed by the penetration of the host cell wall by the tail, or tail-core, of the phage
particle. However, in the case of phage ml 3, penetration of the cell wall must presumably precede attachment of the phage to the plasma membrane. Like many other
phages, phage rnl 3 possesses a muralytic enzyme ( t r a m & Reiter, 1965) which
hydrolyses the linkages between N-acetyl muramic acid and N-acetylglucosamine
residues in the cell wall glycosaminopeptide (tram, 1965). The phage-bound enzyme
has now been shown to lyse-from-without strains of lactic streptococci which do not
adsorb phage ml 3 irreversibly. Hence, although adsorption of phage ml 3 to cell walls
is reversible with respect to phage, the phage may produce holes in the cell wall which
presumably expose the phage receptors in the membrane. It is of interest that Bradley
& Kay (I96O) found that the tail of phage ml 3 (obtained from us but unfortunately
termed 3 ML by them) measured IOOO× 9o A and had no sheath, base plate or other tail
appendage. Base plates and tail fibres are known to be involved in the adsorption of
coliphages T2 and T4 to Escherichia coli (Simon & Anderson, 1967); and staphylococcal
phages, which also adsorb to the host cell wall (Hotchin, Dawson & Elford, 1952)
possess a knob at the base of the tail (Hotchin, 1954; Bradley & Kay, 196o). The tail
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I18
J . D . ORAM A N D B. REITER
o f p h a g e m l 3 was f o u n d b y B r a d l e y & K a y (i96o) to have a cross-sectional d i a m e t e r
o f 90 ~, slightly greater t h a n the 70 ~, d i a m e t e r o f the core o f the s h e a t h e d tail o f colip h a g e T2 (Brenner et aL I959) w h i c h penetrates the o u t e r layers o f the cell wall b u t n o t
the p l a s m a m e m b r a n e o f E . coli ( S i m o n & A n d e r s o n , I967). H e n c e the sheath-less tail
o f p h a g e m l 3 a p p e a r s to be functionally equivalent to t h e tail-core o f the T-even
coliphages, in t h a t each p e n e t r a t e s the cell wall as far as t h e p l a s m a m e m b r a n e .
W e gratefully t h a n k M r s W . Cole for her excellent technical assistance.
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