Isolation and Ultrastructure of Bacteriophages of Group N (Lactic) Streptococci

ZbI. Bakt., 1. Abt. Orig. C 1, 79-91 (1980) Institut fiir Mikrobiologie der Bundesanstalt flir Mi1chforschung, 2300 Kiel, Bundesrepublik Deutschland

Isolation and Ultrastructure of Bacteriophages of Group N (Lactic) Streptococci JURGEN LEMBKE, ULI KRUSCH, ARVED LOMPE, and MICHAEL TEUBER Received August 2, 1979

Summary During a two year screening period, approximately 200 phages active against lactic streptococci have been isolated in a cheese factory from raw milk, cheese whey and starter culture using an indicator set of 24 strains of Streptococcus lactis, 17 strains of S. cremoris and 16 strains of S. lactis subsp. diacetylactis. Electron microscopy of these phages suggests the existence of at least 14 distinct morphological types. The ultrastructural and host range properties of 14 typical phage types are described and discussed. Key words: Bacteriophages - Group N Streptococci - Ultrastructure of phages Introduction Cheese is traditionally produced from milk with the aid of lactobacilli and lactic streptococci. The world-wide manufacture of 1010 kg cheese per year (FAO Production Yearbook, 1977) proceeds under non-sterile conditions. This applies to the substrate milk as well as to the manufacturing equipment. It is therefore not surprising that incidences of phage attacks are common and the main cause of slow and faulty fermentations in the cheese industry (Lawrence et aI., 1976). Although a wealth of information on the occurrence of phages does exist especially in the case of lactic streptococci (recent reviews see Lawrence, 1978), a common basis to compare the data from different laboratories is missing. It is the scope of this publication to describe in detail the ultrastructure of bacteriophages active against Streptococcus lactis, S. lactis subsp. diacetylactis and S. cremoris isolated from a factory during a two year screening period. This description is intended to provide a sound morphological ground for future biochemical, taxonomic and serological studies. Material and Methods 57 strains of S. lactis, S. lactis subsp. diacetylactis and S. cremoris were taken from the culture collection of our institute. They were kept in skim milk at - 82°C. For the use

80

].Lembke, U.Krusch, A.Lompe, and M. Teuber

as phage indicator, M-17 broth (Terzaghi and Sandine, 1975) was inoculated and incubated with the desired strain for 2 h at 30°C. This suspension was stored at 4 °C until used. For the propagation of phages, overnight cultures of streptococci were 1: 20 diluted with fresh M-17 broth containing 10% lactose. Phage infection was after 2 hr growth of the streptococci at 31°C. Isolation of phages. Phages were isolated from raw milk, starter cultures, cheese whey and cheese. 10 ml of raw milk was acidified with 0.3 ml lactic acid (10%) to precipitate casein. Cheese was homogenized and suspended in Ringer solution. Insoluble components were removed by paper filters. The filtrate was made free of bacteria by filtration through membrane filters (pore size 0.45 ,urn). For the enrichment of phages, 0.1 ml of the sterile filtrate of raw milk, whey, cheese and starter cultures were incubated together with 0.1 ml of overnight bacterial cultures in M-17 broth for 10 min and inoculated into 5 ml litmus milk. After 1 hr of incubation at 40°C in a water bath followed by overnight incubation at 30 DC, 0.1 ml was transferred into fresh litmus milk. This transfer procedure was repeated up to 3 times. The content of vials showing inhibition of acid production was filtered through membrane filters (pore size 0.45,um). Purification of phage isolates. Phage types were purified by plaque isolation from dilution series of the sterile filtrate of litmus milk plated on the original sensitive bacterial strain in M-17 broth. The plaque test was performed using the double layer technique (Adams, 1959). Purification of phages for electron microscopy. Although electron microscopy of phages can be performed with crude lysates or directly from plaques in order to identify the morphological type, most of the electron micrographs shown in this publication were obtained from phage preparations purified by cesium chloride or sucrose density centrifugation. Before ultracentrifugation, the phages were enriched from the crude lysates by precipitation with 8% polyethyleneglycol (Yamamoto and Alberts, 1970). Negative staining of phages. Carbon coated mica plates were immersed into the phage containing solution in a manner to allow only a partial floating of the carbon film (Valentine et aI., 1968). The glimmer was then rinsed with dist. water and transferred into the staining solution (0.5-1% uranyl formiate in D 2 0). The floated carbon film was picked up with a freon-washed copper grid (400 mesh). The excess staining solution was removed with filter paper. The grid was immediately transferred into a Philips EM 300 electron microscope. Pictures were taken at a voltage setting of 60 KV and an objective aperture of 50,um on Kodak Fine Grain Release Positive Film 5302 (35 mm). The factor of magnification was determined with the aid of a standard cross grating (d = 0.463,um, Balzers Union, Wiesbaden, FRG). Antisera. Antisera against phages were prepared in rabbits by 4 subcutaneous injections of density gradient purified phages. 1 ml of phage suspension (10" particles), emulsified in 0.5 ml complete Freund's adjuvant was applied at weekly intervals. The rabbits were bled 10 days after the last injection. Immunoelectronmicroscopy. A carbon film was floated on a drop of phage-specific antiserum (1 :500-1 :2000), after 10 min transferred onto a drop of crude whey containing phages (30 min) and again floated on the same antiserum (10 min). After a brief washing on a drop of water the carbon film was transferred to the staining solution and picked up with a 400 mesh copper grid.

Results

1. Isolation and origin of phages Phages were routinely isolated from raw milk, cheese whey and starter cultures of a german cheese factory by testing a set of 57 indicator strains comprised of 17 S. cremoris, 24 S. lactis and 16 S. lactis subsp. diacetylactis strains as described

Bacteriophages of Lactic Streptococci

81

in materials and methods. All phages isolated in our laboratory carry a number with the prefix P given to them in the order of isolation. Additional phages were isolated from an Edam-type cheese produced by the german dairy farm in ChiengMai (Thailand). Some phages were the generous gift of Drs. H.Leesment and ].Dufeu of the Swedish Dairies Association in Malmo (Sweden). Table 1 lists the morphologically distinct phage types discovered in our laboratory in different substrates in the order of their frequency of detection. To avoid a further new nomenclature adding to the existing confusion, we decided to select a type for each morphological class as basis for the ultrastructural description. Of the more than 200 phages isolated 186 were characterized by electron microscopy (Table 1). There is a clear predominance of 4 phage types represented by the type lines P047, POOl, P008 and P109. Whereas the P047 group was mainly detected in raw milk, the other three types dominated in whey. The detection of the same types in raw milk and whey (e.g. type POOl) could indicate raw milk as a source of infection since most types are not completely inactivated by the pasteurization conditions used in Germany (A. Lampe, in prep.). The types mainly isolated from slowly acidifying cheese vats are phages morphologically identical with phages POOl, P008 and P109. The remaining phage types have only been infrequently isolated. 2. Ultrastructure of phages As far as morphological properties are concerned, all isolated phages belong into Bradley's group Band C (Bradley, 1967). The presence of double stranded DNA, however, has only been demonstrated for phage types POOl and P008 (Lembke, Lampe and Teuber, Abstr. XII th Int. Congr. Microbiol. 1978, p. 145) by the melting properties of DNA. Phages having contractile tails have not been observed. In our study the main features allowing a clear morphological distinction and differentiation are head form and size, the presence of a collar, tail length, diameter and structure and the subtile construction of baseplates and fibers. The dimensions of the phages given in Table 1 are mean values measured with up to 30 phage particles. The phages shown in Fig. 1 to 4 may therefore exhibit variations from the mean values described in Table 1. The pictures of negatively stained lactic streptococci phages in Fig. 1 and 2 have been arranged according to the increase in their overall size. For that reason all these phages are depicted at the same magnification. Phage P034 belonging into Bradley's group C is to our knowledge the first description of this type active against lactic streptococci. It has been isolated from raw milk and has also been identified by us in the collection of the Swedish Dairies Association. This phage type is characterized by a short tail showing a spiral shape, a collar extending into at least 4 fibers and a head composed of large capsomers (Fig. 1A; 3A; 3B). The capsomers are especially recognized in the rasp berry appearance of shadowed preparations (Fig.3B). Phages P109 and POOl are very similar with the exception of a missing collar in POOl (Fig. 1B; 1C). Empty heads change their appearance from a prolate into an ovoid form (Fig. 3 C) in both phages. Both phages have a clearly segmented tail with 20 recognizable segments. A small base plate does not show distinct details (Fig. 3D). Phages POB and P059 look very similar in the first view with double collars and massive baseplates (Fig. 1D; 1E; 3E; 3F). Phage P059 has in addition a velamenlike structure around the upper part of the tail (Fig. 1E). 6 Zbl. Bakt. I. Abt. Orig. C 1

82

].Lembke, U.Krusch, A.Lampe, and M. Teuber

Table 1. Characterization of bacteriophages active against lactic streptococci

type strain (host bacterium)

number of isolates

sources of isolation

morphologiCll characterization

P047

51

raw milk (42) whey (7) streptococcus culture (2)

length: 325 ± 8 nm head: 65 nm, isometric; tail: 260 X 14nm; baseplate: 30nm

POOl (S. Tactis subsp. diacetylaetis Bu2)

49

whey (18), raw milk (17) collection Kiel (5) collection Malmo (1) starter culture (3) streptoccocus culture (2) Thai cheese (3)

length: 165 ± 3 nm head: prolate 61 X 44 nm tail: 105 X 11 nm baseplate not distinct

POD8 (S. lactis F7j2)

34

whey (23), streptococcus culture (4), raw milk (3), starter culture (2) collection Kiel (2)

length: 208 ± 2 nm head: isometric 54 nrn, collar present tail: 154 X 12 nm, baseplate not distinct

PI09

24

whey (23) Thai cheese (1)

length: 170 ± 3nm head: prolate 59 X 42 nm, collar present tail: 111 X 11 nm, baseplate not distinct

(S. cremorisWg2)

(S. cremoris K 14/39)

P013 (S. lactis subsp. diacetyTactis Bu2)

6

whey (3), raw milk (1) collection Kiel (1) collection Malmo (1)

length: 183 ± 5 nm head: isometric 55 nm, double collar tail: 128 X 11 nm baseplate large, 25 nm

P219

4

whey (4)

length: 206 ± 3 nm head: isometric 52 nm, collar present tail: 154 X 13 nm, baseplate nJt distinct

P034 (S. lactis "MLl")

4

raw milk (3) collection Malmo (1)

length: 89 ± 2 nm head: prolate 65 X 44 nm, collar present tail: short, 24 X 10 nm

P026 (S. lactis Est5)

3

whey (1), raw milk (1) collection Malmo (1)

length: 580 ± 25 nm head: isometric 80 nm tail: 500 X 12.5 nm baseplate not distinct

PI07 (S. lactis subsp. diacetyTactis R2Ajl)

2

whey (2)

length: 207 ± 6 nm head: isometric 55 nm tail: 152 X 11 nm, long tail fiber baseplate not distinct

P142

2

raw milk (1) colleCtion Malmo (1)

length: 199 ± 4nm head: isometric 52 nn1, collar present tail: 147 X 10.5 nm, baseplate 26 nm

P087 (5. lacris subsp. diacetylactis "e 13")

raw milk

length: 265 ± 5 nm head: isometric 65 nm tail: 200 X 16 nm baseplate large, 40 nm

P204 (S. lactis 8)

collection Malmo

length: 310 ± 7 nm head: isometric 55 nm tail: 255 X 10 nm, baseplate 16 nm

P059

whey

like P013 double collar present additional collar structure

Thai cheese

length: 199 ± 5 nm head: isometric 49 nm, collar present tail: 150 X 14 nm, baseplate not dist.

(S. cremoris K20j21)

(S. cremoris F22)

(S. cremorisWg2)

Pl91 (5. Tactis R 12Ajl)

Bacteriophages of Lactic Streptococci

83

Fig. 1. Electron micrographs of negatively stained bacteriophages active against lactic streptococci. (A) phage P034; (B) P109; (C) POOl; (D) P013; (E) P059; (F) P19l; (G) P 008; (H) P 219; (I) P 142. The factor of magnification is the samefor all pictures (see bar).

Phages P191 and P008 are again very similar. However, P191 has a thicker tail which seems not to be segmented (Fig. IF). It has a drill-like base (Fig. 3G) POD8 reveals a segmented tail with a knob-like base (Fig 1G). Phage P 219 is mainly distinct from P 191 and POD8 by it's differentiated baseplate demonstrating a hexagonal array at the tip of the tail (Fig. 1 H). Phage P142 is distinguished by a very complex, double baseplate (Fig. 31) with attached swollen "fingers" (Fig. 11; 3H). Sometimes a spike seems to emerge between the "fingers" in the middle axis of the tail (Fig. 3H, right tail tip) Phage PD47, common in raw milk, reveals a sixfold radial symmetry of the baseplate (Fig 2A; 4 E; 4 F) similar to P219. It is not possible to resolve more clearly the obviously segmented structure of the tail.

84

J. Lembke, U. Krusch, A. Lompe, and M. Teuber

Fig. 2. Electron micrographs of negatively stained bactetiophages active against lactic streptococci (continued from Fig. 1). (A) P047; (B) P087; (C) P107; (D) P204; (E) P026. The factor of magnification is the same as in Fig. 1.

Phage P087 is characterized by a typical and specific, possibly helical structure of its tail (Fig. 2B; 4 B; 4 C). The shape of the empty head (Fig. 4 A) may suggest an octahedral structure A complex baseplate completes the interesting morphology of this phage (Fig. 4B; 4C). Phage P 107 has an unique long tail fiber originating from a small baseplate (Fig.2C).

Bacteriophages of Lactic Streptococci

85

Fig. 3. Electron micrographs of detailed structures of negatively stair.ed and of shadowed phages of lactic streptococci. (A) P034 negatively stained; (B) P034 shadowed; (C) POOl negatively stained; (D) tip of tail P109; (E, F) tips of tails POB; (G) tips of tails P19l; (H, I) tips of tails P 142. The unmarked bars represent 50 nm.

86

J.Lembke, U.Krusch, A.Lompe, and M.Teuber

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~

o

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Fig. 4. Electron micrographs of detailed structures of negatively stained phages of lactic streptococci (continued from Fig. 3). (A) empty head P08?; (B, C) tips of tails P08?; (D) empty head P04?; (E, F) tips of tails P04?; (G) empty head P026; (H) tip of tail P026; (I) tips of tails P204. The unmarked bars represent 50 nm.

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J. Lembke, U. Krusch, A. Lompe, and M. Teuber

Phage P 204 has only been identified in the Malmo collection It possesses an unusually long, well resolved and segmented tail The basis of the tail may extend into several delicate, finger-like appendices (Fig. 2D; 41). Phage P026 belongs to largest phages ever described for lactic streptococci The flexible and long tail is clearly segmented (Fig. 2E). A distinct baseplate cannot be demonstrated (Fig. 4H). The empty head is similar to that of P087. 3. Host range of the types of phages Table 2 shows the host range of most of the investigated phage types. The indicated bacterial strains have been used for our screening programme. The scheme is mainly intended to demonstrate that almost all phages do cross the species boundaries of lactic streptococci. We will prove in a forthcoming publication that the host range of individual phage types within one morphological group may vary to quite an extent. Phages P219, P204 and Pl07 have been isolated so recently that a complete host range is not yet available.

Discussion The problem of phage isolation is mainly a matter of the availability of appropriate indicator bacteria. We solved this problem by using a large set of bacterial strains from our collection containing starter strains used in the screened factory and a number of internationally renowned strains e. g. of the C-series and NCDO (National Collection of Dairy Organisms). The second problem is the taxonomic identification of these strains as S. lactis, S. lactis subsp. diacetylactis and S. cremoris. All strains indicated in this publication have been carefully reinvestigated in this respect using common classification properties like arginase reaction, citrate utilization, salt tolerance and so on (Bergey, 1975) and growth properties on the differentiating medium of Reddy et aI. (Reddy et aI., 1972). In accordance with Lawrence, (Lawrence, 1978) we observe a variability within our bacterial cultures leading to a reclassification from one into another species. It must be the main goal of dairy microbiologists to make clear the reasons for this variability and to develop a concept of the species definition within the lactic streptococci. During the course of this work, it turned out to be extremely important to control the purity of phage isolates by electron microscopy since serial plaque purification did not always produce pure isolates but mixtures of 2 or 3 phage types. Especially the raw milk phage type P047 exhibiting pin point plaque morphology can be easily transferred as a contaminant. This can happen if a phage with large plaques e.g. POOl and a similar host range is to be purified. The difficulty to compare the ultrastructure of bacteriophages of lactic streptococci described in the literature comes from the different techniques employed to prepare and stain the phages for electron microscopy. Sometimes, the resolution of the published micrographs is not high enough to allow an interpretation with regard to morphological details elaborated in our study. In principle, the main morphological types known from the literature have all been identified in our work (Bauer et aI., 1970; Jarvis, 1977; Lawrence, 1978; Nyiendo, 1974; Terzaghi, 1976; Keogh, 1974; Tsaneva, 1976; Engel et aI., 1975). This proves that our set of 57

Bacteriophages of Lactic Streptococci

89

indicator bacteria has been sufficient. Future ultrastructural work will have to run parallel with biochemical, host range and immunological studies. The immunological characterization can be rapidly achieved with very small amounts of antigen and antisera by immunoelectron microscopy (Fig. 5). The only phage type not yet

100nm

Fig. 5. Immunoelectron micrograph of phage P008. The negatively stained preparation shows agglutination of the phages and a dense fur of antibodies at the whole phage surface (compare Fig. 1 G). Use of an heterologous antiserum did not produce these effects. 10 4 -10" phages/ml can be detected by this technique.

90

J. Lembke, U. Krusch, A. Lompe, and M. Teuber

reported in lactic streptococci is phage P034 belonging into Bradley's group C (Bradley, 1967). The ultrastructural diversity evident from the work of other laboratories and our own does in our opinion not justify the view of Lawrence (Lawrence, 1978) that large isometric and prolate phages are mutants of small headed isometric phages isolated from lysogenic streptococci. The biochemistry and molecular biology of lactic streptococci will only be completely understood if phage sensitivity, lysogeny and plasmid pattern are simultaneously studied with a representative set of strains. The future use of these bacteria in a highly computerized and automated food industry will function it a proper management of lactic streptococci can be developed.

Acknowledgements We thank U.Leben for her excellent technical assistance, B.Fahrenholz for his help with electron microscopy and Dr. G. Engel for the gift of some bacteriophages isolated in his previous study (Engel et a!., 1975).

References

Adams, M. H.: Bacteriophages. New York, Interscience Publishers, Inc. 1959 Bauer, H., Dentan, E., Sozzi, T.: The morphology of some streptococcus bacteriophages. J. Microsc. 9, 891-898 (1970) Bergey's Manual of Determinative Bacteriology, Eighth Edition, Baltimore, The Williams and Wilkins Company (1975) Bradley, D.E.: Ultrastructure of bacteriophages and bacteriocins. Bact. Rev. 31, 230-314 (1967) Engel, G., Milczewski, K.E. von, Lembke, A.: Versuche zur Differenzierung von Phagen der Streptococcus lactis- und cremoris-Gruppe: Bakterienspektrum, serologische Beziehungen und morphologische Kriterien. Kieler Milchwirtsch. Forschungsber. 27,25-48 (1975) FAO Production Yearbook, Vo!. 31. Rome (1977) Jarvis, A. W.: The serological differentiation of lactic streptococcal bacteriophage. N.Z.]. Dairy Sci. Techno!. 12, 176-181 (1977) Keogh, Barbara R., Shimmin, P. D.: Morphology of the bacteriophages of lactic streptococci. App!. Microbio!. 27,411-415 (1974) Lawrence, R. C.: Action of bacteriophage on lactic acid bacteria: consequences and protection. N.Z.J. Dairy Sci. Techno!. 13, 129-136 (1978) Lawrence, R. C., Thomas, T. D., Terzaghi, B. E.: Reviews of the progress of Dairy Science: Cheese starters. J. Dairy Res. 43,141-193 (1976) Nyiendo, J. A.: Studies on host range, fine structure, and nucleic acids of lactic streptococcus bacteriophages. Thesis, Oregon State University, 1974 Reddy, M.S., Vedamuthu, E.R., Washam, C.]., Reinbold, G. W.: Agar medium for differential enumeration of lactic streptococci. App!. Microbiol. 24, 947-952 (1972) Terzaghi, B. E.: Morphologies and host sensitivities of lactic streptococcal phages from cheese factories. N.Z.J. Dairy Sci. Techno!. 11, 155-163 (1976) Terzaghi, RE., Sandine, W.E.: Improved medium for lactic streptococci and their bacteriophages. Appl. Microbiol. 29, 807-813 (1975) Tsaneva, K.P.: Electron microscopy of virulent phages for Streptococcus lactis. App!. Environm. Microbio!. 31, 590-601 (1976)

Bacteriophages of Lactic Streptococci

91

Valentine, R. C., Shapiro, R. M., Stadtman, E. R.: Regulation of glutamine synthetase. XII. Electron microscopy of the enzyme from Escherichia coli. Biochemistry 7, 2143 (1968)

Yamamoto, K.R., Alberts, R.M.: Rapid bacteriophage sedimentation in the presence of polyethylene glycol and its application to large-scale virus purification. Virology 40, 734-744 (1970) Prof. Dr. M. Teuber, Institut flir Mikrobiologie der Bundesanstalt fur Milchforschung, Hermann-Weigmann-Str. 1-27, D-2300 Kiel