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APPLIED MICROBIOLOGY, Feb. 1974, p. 411-415Copyright @ 1974
American Society for Microbiology
Vol. 27, No. 2Printed in U.SA.
Morphology of the Bacteriophages of Lactic StreptococciBARBARA
P. KEOGH AND P. D. SHIMMIN
Division of Food Research, Dairy Research Laboratory, CSIRO,
Melbourne, Australia
Received for publication 5 September 1973
Electron microscope studies have been made of a number of phages
of lacticstreptococci, seven of which were phages of Streptococcus
lactis C10. Two of thephages are thought to be identical; five have
been classified by the method ofTikhonenko as belonging to group IV
(phages with noncontractile tails) with typeIll tail plates; one
belongs to group V (phages with tails possessing a
contractilesheath). Both prolate polyhedral heads and isometric
polyhedral heads arerepresented among the group IV phages. The
phage drc3 of S. diacetilactis DRC3has been shown to have similar
structure to the group IV phages of S. lactis C10with prolate
polyhedral heads. The phages mll, hp, cll, and z8 of the S.
cremorisstrains ML1, HP, C11, and Z8, respectively, were shown to
belong to the group IVphages with type III tail plates by the
method of Tikhonenko. All had octahedralheads and tended to be
larger than most of the other phages studied.
Several studies of the morphology of thebacteriophages of the
lactic streptococci havebeen made within the last two decades (8,
9,12). In recent years, they have received rela-tively little
attention despite the considerableadvances in the techniques of
electron micros-copy. Bradley and Kay (4) studied a phage
ofStreptococcus lactis 3ML with a prolate headwith no sharp angles
and reported it to be of ashape not described previously. Later,
Bradley(2) demonstrated head dimorphism of thisphage. Tikhonenko
(10), in her book, has in-cluded electron micrographs and
descriptions ofthree phages of S. lactis. Bauer, et al (1)
havestudied perhaps the widest range of phages oflactic
streptococci. Their studies included thephages of S. lactis, S.
thermophilus, S. diaceti-lactis, and S. cremoris. McKay and Baldwin
(7)have published electron micrographs of a phageproduced by the
ultraviolet light irradiation of alysogenic culture of S. lactis
C2.This paper describes the morphology of some
phages of S. lactis, S. diacetilactis, and S.cremoris
strains.The strain differentiation of the lactic strep-
tococci used as single-strain cheese starters isbased on their
susceptibility to phage; manydifferent phages for each different
strain withineach species have been isolated. Seven phagesdescribed
in this paper share the host S. lactisC10. It is possible that a
knowledge of themorphology of the phages, particularly thosewith a
common host, could help in understand-ing how new phages arise in
the cheese factoryenvironment.
MATERIALS AND METHODSAll the phages studied were isolated by
members of
the staff of this laboratory during the period 1955 to1972. The
phages of S. lactis C10 were c10I, c10I1,c10III (5), 10p, 10n, 106.
and l0w. Phage 106 is alsoknown as c6 and will attack both S.
lactis strains C6and C10.
Other phages studied are as follows: c2, a phage ofS. lactis C2;
drc3, a phage of S. diacetilactis DRC3;mll, a phage of S. cremoris
ML1; hp, a phage of S.cremoris HP; cli, a phage of S. cremoris C11;
and z8,a phage of S. cremoris Z8.
Concentration of phages and electron micros-copy techniques.
Phage concentrations were pre-pared after the lysis of host strains
of lactic strepto-cocci on plates which were prepared by the
double-layer plate method by using the media described byKeogh and
Pettingill (6) and incubating overnight.An inoculum of phage known
to give confluent lysiswas used in preparing the plates. The
surface layerwas removed, broken up, washed with 0.1 M ammo-nium
acetate, and centrifuged at approximately 5,000x g for 30 min. The
supernatant was then centrifugedat 120,000 x g for 1 h. Grids were
prepared from thedeposit and negatively stained with neutral 2%
(wt/vol) potassium phosphotungstate.
Electron micrographs were taken with a Siemenselectron
microscope model Elmiskop I at a magnifi-cation of x40,000.
RESULTSIn the interpretation of the results, the classi-
fication of the phages by Tikhonenko (10) wasfollowed. This
applies also to the typing of thetail plates according to their
morphology.Table 1 shows the dimensions of the phages
studied. The various phages for which S. lactis411
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KEOGH AND SHIMMIN
TABLE 1. Dimensions of lactic streptococcus phages
Hedd-Tail dimensions Type of lsi-Host strain Phage Shape of head
mensionsLegh WidthTp of tailpaenm pltail cation"
(n) Type of tail (group)(non) (non) (type)
S. lactisC10 c10I Prolate poly- 51 by 45 92 5 Noncontractile 10
III IV
hedralC10 c10dI Prolate poly- 55 by 42 92 9 Noncontractile 12
III IV
hedralC10 c10III Octahedral 70 by 70 180 12 Contractilec 30
vc
(with spreadfibers, 50)
C10 lop Octahedral 55 by 55 127 5 Noncontractile 10 III IVC10
iOn Octahedral 55 by 55 127 5 Noncontractile 10 III IVC10 106 (c6)
Prolate poly- 55 by 40 92 10 Noncontractile 15 III IV
hedralC10 low Prolate poly- 62 by 45 90 7 Noncontractile 10 III
IV
hedralC6 106 (c6) Prolate poly- 55 by 40 92 10 Noncontractile 15
III IV
hedralC2 c2 Prolate poly- 55 to 62 102 9 Noncontractile 10 III
IV
hedral by 45
S. diacetilactisDRC3 drc3 Prolate poly- 56 by 45 102 9
Noncontractile 16 III IV
hedralS. cremorisML1 mol Octahedralc 60 by 60 155 12
Noncontractile 22 III IVHP hp Octahedralc 50 by 50 165 10
Noncontractile 17 III IVC011 cl Octahedralc 75 by 75 132 14
Noncontractile 17 III IVZ8 z8 Octahedralc 60 by 60 162 10
Noncontractile 17 III IV
a Including tail plate.0 By the classification method of
Tikhonenko (10).c Not assigned with certainty.
C10 is the host differ in their ultrastructurefrom each other.
Phages clOI, clOII, and 106,however, are very similar in
morphology, show-ing only minor differences in dimensions of
themain structures (Fig. 1A, B, F). Although inburst size
experiments phage 106 was greatlyinfluenced by the host strain
(5a), it shows thesame morphology irrespective of the host (C6
orC10) on which it is grown (Fig. 1F, Fig. 2A).Phage clOII is
morphologically distinct fromall the other phages included in these
studies.Although no contracted sheath was seen, itstail has the
characteristic end plate and tailfibers of a phage possessing a
contractilesheath (Fig. 1C). It belongs, therefore, to groupV
according to the classification of Tikhonenko(10). Phage lOw is a
group IV phage with a pro-late polyhedral head, much larger than
theothers of the same shape described above (Ta-ble 1, Fig.
1G).Phages lOp and iOn have noncontractile tails;
but whereas those described above having non-contractile tails
have prolate polyhedral heads,lOp and iOn have octahedral heads and
longerand narrower tails (Table 1, Fig. 1D, E).Phage c2 of another
strain of S. lactis, C2, was
studied (Fig. 2B). It has an elongated head anda noncontractile
tail; in addition, it has awell-defined collar. Heads of different
lengthscan be seen in the electron micrographs (Table1), but there
is not a well-defined dimorphismas described by Bradley (2). In
shape this phageis unlike the temperate phage of S. lactis
C2studied by McKay and Baldwin (7), the latterhaving an isometric
polyhedral head. The onlyfeature the two phages have in common is
thepossession of a well-defined collar.Phage drc3 of S.
diacetilactis DRC3 (Fig. 2C)
has been shown to have similar structure to thegroup IV phages
of S. Iactis C10 with prolatepolyhedral heads.The phages of the S.
cremoris strains ML1,
HP, C11, and Z8 studied here (Fig. 2D, E, F, G)have isometric
polyhedral heads which appearto be octahedral. They cannot be
assigned withcertainty however. Phages cli and mll tend tohave a
much larger head than all others shown,whereas hp has a much longer
tail (with theexception of clOIII). All four S. cremoris
phagesstudied belong to group IV, by the classificationof
Tikhonenko (10), and have type III tailplates.
412 APPL. M1CROBIOL.
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FIG. 1. Electron micrographs of some bacteriophages which attack
Streptococcus lactis C10. Magnificationx2OO,OOO; 1 cm = 50 nm. (A)
Phage c1I, (B) phage clOII, (C) phage c1OIII, (D) phage 10p, (E)
phage iOnshowing isolated tail plates (arrows), (F) phage 106 grown
on C10, and (G) phage 10w.
413
F
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,t
i.
w' t.
_ , 4.,¢ ..mllA'
C
F- . - ,_ t o -e.FIG. 2. Electron micrographs of some
bacteriophages of Streptococcus lactis, S. diacetilactis, and
S.
cremoris strains. Magnification x200,OOO; 1 cm = 50 nm. (A)
Phage 106 grown on C6, (B) phage c2, (C) phagedrc3, (D) phage mll,
(E) phage hp, (F) phage clI, and (G) phage z8.
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PHAGES OF LACTIC STREPTOCOCCI
DISCUSSIONIt is possible that there are, in other laborato-
ries, phages bearing the same name yet havingdifferent
morphology from those described. Thisconfusion arises (5a) because
of the prevailinghabit in the field of dairy microbiology of
nam-ing phages after the lactic streptococcal strainswith which
they were first found to be associ-ated. In this paper, the
original names havebeen kept. A different code, however, wouldbe
preferable in the naming of new isolates. Inthe future, it may be
advisable to be guided bythe decisions of the recently formed
Interna-tional Committee on Nomenclature of Viruses(11). Present
studies of the ultrastructure andother characteristics of the
phages of S. lactisC10 (5, 5a) have shown that six of the
sevenphages appear to be different. Phages c10I,c10dl, and 106 are
very similar in morphology,showing differences, albeit, only in
measure-ment. One can speculate that these phagescould have arisen
by mutation from a parentstrain. On the other hand, c10III is a
very dif-ferent phage in its ultrastructure and probablyhas a
different genetic origin.
It is thought that 10n and 10p are temperatephages and are, in
fact, the same phage strain,because they have the same morphology
andboth were isolated under identical conditions offailure of the
continuous culture of S. lactis C10in separate laboratories. Their
temperate na-ture, however, has not been proved. Theirplaque
morphology is consistent with that of atemperate phage.The phages
of the strain of S. cremoris so far
studied by electron microscopy are very similarin
ultrastructure, varying only in the size of thecomponent
structures. They all belong to groupIV.This study has shown that
the phages of
lactic streptococci investigated here can beclassified, by the
method of Tikhonenko (10),into groups IV and V, corresponding
toBradley's (3) group B and A, respectively.Within these
classifications, the lactic strepto-coccal phages studied here are
morphologicallydistinguishable from each other.
LITERATURE CITED
1. Bauer, H., E. Dentan, and T. Sozzi. 1970. The morphol-ogy of
some streptococcus bacteriophages. J. Microsc.9:891-898.
2. Bradley, D. E. 1963. The structure of some Staphylococ-cus
and Pseudomonas phages. J. Ultrastruct. Res.8:552-565.
3. Bradley, D. E. 1967. Ultrastructure of bacteriophages
andbacteriocins. Bacteriol. Rev. 31:230-314.
4. Bradley, D. E., and D. Kay. 1960. The fine structure
ofbacteriophages. J. Gen. Microbiol. 23:553-563.
5. Czulak, J., and J. Naylor. 1956. Host-phage relationshipof
cheese starter organisms. Part 1. J. Dairy Res.23:120-125.
5a. Keogh, B. P. 1973. Adsorption, latent period and burstsize
of phages of some strains of lactic streptococci.J. Dairy Res.
40:303-309.
6. Keogh, B. P., and G. Pettingill. 1966. Long-term storageof
bacteriophages of lactic streptococci. Appl. Micro-biol.
14:421-424.
7. McKay, L. L., and K. A. Baldwin. 1973. Induction ofprophage
in Streptococcus lactis C2 by ultravioletirradiation. Appl.
Microbiol. 25:682-684.
8. Parmelee, C. E., P. H. Carr, and F. E. Nelson. 1949.Electron
Microscope studies of bacteriophage activeagainst Streptococcus
lactis. J. Bacteriol. 57:391-397.
9. Sandine, W. E., P. R. Elliker, and H. A. Hays.
1960.Bacteriophage-lysis of Streptococcus diacetilactis andits
effect on biacetyl production in mixed-strain startercultures. J.
Dairy Sci. 43:755-761.
10. Tikhonenko, A. S. 1970. Ultrastructure of bacterial
vi-ruses. Plenum Press, New York.
11. Wildy, P. 1971. Classification and nomenclature of vi-ruses,
p. 12-15. In Monographs in virology, vol. 5. S.Karger, Basel.
12. Williamson, K. I., and W. S. Bertaud. 1951. A
newbacteriophage active against a lactic streptococcus.
J.Bacteriol. 61:643-645.
415VOL. 27, 1974
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