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.

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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.

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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.

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