Morphology of phages of a general Salmonella typing set

Res. Virol. 1992, 143, 303-310

© INSTITUTPASTEUR/ELSEVIER Paris 1992

Morphology of phages of a general Salmonella typing set H.-W. A c k e r m a n n 0)(') and M. Gershman (2)

(0 Fdlix d'Hdreile Reference Center for Bacterial Viruses, Department of Microbiology, Faculty of Medicine, Laval University, Quebec, Qc, GIK 7P4 (Canada), and (e) Department of Biochemistry, Microbiology, and Molecular Biology, University of Maine, Orono, ME 04469-0131 (USA)

SUMMARY The typing set includes 27 tailed phages belonging to three families, the Myoviridae (contractile tails, 4 phages), Siphoviridae (long, non-contractile tails, 21 phages) and Podoviridae (short tails, 2 phages). Heads are isometric or elongated. The phages fall into 10 morphological groups, seven of which correspond to known enterobactedal phage species (Jersey, N4, T5, T7, ZG/3a, X, 16-19) and one to a Rhizobium phage species (CM O. TS-type and T7-type phages are for the first time reported in salmonellae. Two phages produce large amounts of abnormal mottled heads.

Key-words: Bacteriophage, Salmonella, Typing; Morphology, Taxonomy. INTRODUCTION Phage typing schemes have been proposed for a b o u t 25 Salmonella serovars (Gershman, 1976a,b; for reviews, see A c k e r m a n n and DuBow, 1987a; and Kasatiya and NicoUe, 1978). They are of proven epidemiological value and often allow tracing epidemics to theL origins, but are applicable to individual serovars only. One of us has developed a set of 50 phages, later reduced to 27, for the whole genus Salmonella (Gershman, 1977; Gershman and Markowsky, 1983). The set is applicable to at least 86 Salmonella serovars corresponding to about 95 ¢/0 of isolates. The phages of this set have not been investigated and are essentially unknown. A study of their morphology is desirable for comparison with other enterobacterial phages and for purposes of identity control during propagation.

MATERIALS AND Mi~THODS Bacteriophages Phages were isolated from sewage in the years 1970-72 by the enrichment method (Gershman, 1977) and propagated on strains of S. anatum, S. binza, S. enteritidis, S. heidelberg, S. newport, S. senftenberg and S. thompson (Gershman and Markowsky, 1983). Among the phages of the present set, phages 8, 16, and 26 were from water samples from New Y6rk State, North Carolina, and Florida, respectively. The remainder were from various localities in Maine. Electron microscopy Lysates titering 107 to 108 PFU/ml were filtered through membrane f'fltersof 0.45 tan (Millipore Corporation, Bedford, MA). Phages were sedimented at 70,000 g for 60 rain using a Beckman (Palo Alto, CA) L8M ultracentrifuge and a SW50.1 rotor. Alternatively, a Beckman J2-21 centrifuge and a JA-21

Submitted May 21, 1992, accepted September 10, 1992. (*) Vice-chairman,BacterialVirusSubcommitteeof InternationalComitteeon Taxonomyof VirusesOCTV),and correspondingauthor.

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rotor were used at 30,000 g for 60 rain. Pellets were resuspended and washed twice in TAM buffer (sodium azide, 10 mM; Tris-HCl, 10 mM; MgSO4, 1 raM; pH 8.0) (Hohn et al., 1979). Purified phages were deposited on copper grids provided with carbon-coated Formvar films, stained with uranyl acetate (2 ¢/0, pH 4.0) or potassium phosphotungstate (2 %, pH 7.2) and were examined in a "Philips EM 300" electron microscope. Magnification was monitored with catalase crystals (Worthington, Freehold, NJ) (Luftig, 1967). Only negatively stained phages were measured. RESULTS

The phages are tailed and belong, according to gross morphology, to the Myoviridae, Siphoviridae and Podoviridae families (Francki et al., 1991) and to five morphotypes (A1, A3, B1, B2, C1) (Ackermann and Eisenstark, 1974). Heads are isometric or elongated and tails are contractile, long and non-contractile, or short. Most phages can be related by their morphology to well-known species of enterobacterial phages (table I) and two of them correspond to a Rhizobium phage species. Descriptions of these species and references may be found in a taxonomical review (Ackermann and DuBow, 1987b). Isometric heads are icosahedra as indicated by the simultaneous observation of capsids with hexagonal and pentagonal outlines (fig. la-c). Capsid walls are about 3 nm thick. The tail width is 16-18 nm in contractile tails and 7-11 nm in non-contractile tails, respectively. Long tails show 3-4-rim wide transverse striations and inner channels of 2-3 nm in width, which are visible on empty particles only. The phages present common staining artifacts already described in the literature (Ackermann and DuBow, 1987a). For example, large phage heads often appear rounded and flattened after phosphotungstate (PT) staining. Heads stained with uranyl acetate (UA) appear much more regular and angular in outline, but are sometimes positively stained and appear then as deep black, shrunken (fig. 2c), and usually surrounded by a halo which increases with exposure to the beam. Long non-

PFU

-- plaque-forming unit.

contractile tails are relatively thin and flexible in PT and more thick and rigid in UA. Except for positively stained heads, dimensions of UAstained particles are considered as more reliable than those measured after PT staining. The main dimensions of phages are listed in table I. The phages may be described as follows:

1) Myoviridae (contractile tails, 4 phages) Phages 15 and 17 have heads and belong to morphotype A1 (fig. la-c). Heads tend to take up UA and to stain positively. Tails are rigid and have a neck provided with a tiny disk or collar, a base plate, and club-shaped terminal projections. The phages are morphologically identical and indistinguishable from Rhizobium phage species CM l (Werquin et al., 1988). Their characteristic morphology suggests some, possibly remote, phylogenetic relationships between enterobacteria and rhizobia. Morphotype A3 includes phages with elongated heads and is represented by phages 11 and 12 (fig. 2a-c), both members of Salmonella phage species 16-19.

.2) Siphoviridae (long, non-contractile tails, 21 phages) Morphotype B1, characterized by isometric heads, includes 20 phages belonging to five species. Phages 21 and possibly 24 represent new species. a) Phage 21 (fig. 3a-c) is very large and has a fragile head which ruptures easily in phosphotungstate. Its tail is more or less rigid and pointed, shows about 60 transverse striations and has a single, 12 nm long terminal fiber. In addition to normal panicles, lysates contain numerous aberrant, mottled heads. They are either of normal shape and size and may then be provided with a tail, or are irregular in outline and resemble •small polyheads. Similar structures, first observed in phage T4 (Aebi et al., 1976), have been reported in a growing number of phages of widely different bacterial genera (Ackermann

M O R P H O L O G Y OF GENERAL S A L M O N E L L A P H A G E TYPING SET

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Fig. 1. Phage 15 with extended tail. Capsids show edges (a), uranyl acetate uptake with partial positive staining (b), and pentagonal outlines (c). Collars are indicated by arrows.

Fig. 2. Phage 11 with extended tail. Capsids are rounded and slightly flattened (a) or show parallel sides and an angular outline Co) or positive staining and shrinkage (c).

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Table 1. Taxonomic position and main dimensions of phages. Uranyl acetate Morphotype

Phages

AI A3 Bl

15, 17 11, 12 21 l 2, 4, 6, 9, 13, 14, 16, 18, 19, 20, 22, 23, 25, 26 24 3, 7, 8 27 l0 5

B2 C1

Species

Phosphotungstate

N

Head

Tail

N

Head

Tail

× T5

20 30 20 20 140

88 I02 x45 76 62 78

114 104 268 226 195

20 20 20 20 20

88 102x 57 84 65 75

110 103 260 227 190

), ? Jersey ZG/3A N4 T7

20 30 20 20 20

64 60 70 x 53 59 59

175 ll9 180 11 Il

20 20 20 20 20

67 57 73 x 55 61 58

175 113 178 Il 11

16-19

N = Number of particles measured,nm= nanometers. Diametersof isometricheads are betweenopposite apices.

and DuBow, 1987a), suggesting that their production, accidental or not, is a general feature o f tailed phages. By its outstanding tail length, phage 21 differs from any enterobacterial phage k n o w n and is considered as representative o f a new phage species. b) Eighteen phages belong to known species. Phage 1 (fig. 4) is a m e m b e r o f species )~, which lyses flagellated strains o f Escherichia coli and the genera Salmonella and Serratia. It may be noted that this species, while morphologically homogeneous, is divided into three DNA homology groups (F. Grimont, Pasteur Institute, Paris, personal communication). Phage 2 and 13 other isolates (fig. 5) are attributed to species T5. Phages o f this type have so far not been reported in salmonellae. Our phages seem to have significantly smaller heads than phage T5, whose

head diameter has recently been given as 90 n m ( M c C o r q u o d a l e and Warner, 1988). However, in reexamining the T5 head under careful magnification control, we always find diameters o f about 78 nm. Larger diameters are probably due -to flattening. Finally, phages 3, 7, and 8 (fig. 7) are attributed to species Jersey. As in phage Jersey itself, lysates contain a few particles with abnormally long tails. c) Phage 24 (fig. 6) is lambdoid in appearance, but has a longer tail than ), (175 vs. 150 nm) and at least one short fiber o f 23 × 2 nm. It m a y derive f r o m phage ;k by elongation o f the tail. Although clearly different from the other phages studied here, phage 24 is, in the absence o f m o r e data, best not classified at the time being.

Fig. 3. Phage 21. A normal phage and an empty head are seen in (a). Mottled heads are numerous and hexagonal or irregular in outline (b), and may even have a tail (c, arrow). Fig. 4. Phage 1. The tail is thick and tapering and displays a single curled tail fiber.

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MORPHOLOGY OF GENERAL SALMONELLA PHAGE TYPING SET M o r p h o t y p e B2, which features elongated heads, is represented by a single isolate, phage 27 (fig. 8). The phage is attributed to a series of poorly described, possibly heterogeneous phages, designated as species Z G / 3 A . As in phage 21, preparations contain large numbers of mottled heads o f various sizes and shapes, which are sometimes provided with a tail. 3) Podoviridae (short tails, two phages of the C1 morphotype)

Phage 10 (fig. 9) is morphologically related to coliphage N4. Phages of this type, characterized by spikes on both sides of the tail, are frequent in E. coli and Klebsiella bacteria. Many of them are capsule-specific (Ackermann and DuBow, 1987b). Phage 5 (fig. 10) closely resembles coliphage T7 in size and the pointed aspect of its tail. This is the first observation of T7-1ike phages in the genus Salmonella. DISCUSSION Out of a total of about 4,000 phages studied by electron microscopy, no less than 673 are

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tailed phages of enterobacteria (Ackermann, 1992) and at least 30 phage species are recognizable among the latter (Ackermann and DuBow, 1987b). In view of this considerable a m o u n t of observations, it is surprising to find anything new in enterobacterial phages. Nonetheless, at least two new phage species are reported here. Phages 15 and 17, although they have an equivalent among R h i z o b i u m phages, have so far not been observed in enterobacteria. The new species is named Senl5 after the propagating host of these phages, S. enteritidis (Gershman and M a r k o w s k y , 1983). The other species is represented by phage 21, which exceeds any enterobacterial phage in size and resembles some large Bacillus and Streptomyces phages. This species is named S'an21 after its usual host, a S. anatum strain. Phage 24 may represent a further new species or, alternatively, a size variant of lambdoid phages. It is also interesting that T5-1ike and T7-1ike phages are not confined to E. coil and its close relatives, and occur in salmonellae as well. Clearly, even extensively studied phage groups may reveal surprises and the variety of bacterial viruses in nature can only be guessed.

Fig. 5. Phage 2 with flexuous tall and at least three tall fibers. The head is slightly wavy in outline, indicating the presence of capsomers.

Fig. 6. Phage 24 with tapering tail and tail fiber. Fig. 7. Phage 3 with rigid tail and conspicuous base plate. Fig. 8. Phage 27. An empty particle, an intact virion and an irregular mottled head are shown. The empty phage head has collapsed. Fig. 9. Phage 10 with multiple fall fibers. Fig. 10. Phage 5 with tapering tall.

Figs. la to lc, 2b, 2c, 3a, 3c, 4, and 7, uranyl acetate; figs. 2a, 3b, 5, 6, 9, and 10, phosphotungstate. Final magnification, x 297,000 except for fig. 3b (x 148,500); reduced by a factor of 1/3. Bars indicate I00 nm.

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Morphologie des bact6riophages de Salmonella ~ patlir d'un sch6ma de lysotypie Le sch6ma de lysotypie comprend 27 bacteriophages caud6s appartenant/~ trois families, les Myoviridae (queues contractiles, 4 phages), Siphoviridae (queues longues et non-contractiles, 21 phages) et Podoviridae (queues courtes, 2 phages). Les t~tes sont isom6triques ou allong~es. Les phages sont divis6s en 10 groupes morphologiques dont sept correspondent /~ des esp~ces phagiques connues chez les ent6robact~ries (Jersey, N4, T5, T7, ZG/4A, X, 16-19) et un une esp6ce phagique de Rhizobium (CMI). Des phages des types T5 et T7 sont pour la premiere fois d~cdts chez les salmonelles. Deux phages produisent de grandes quantit6s de t~tes anormales tachet~es. Mots-cl~s : Bact6riophage, Salmonella, Lysotypie; Morphologie, Taxonomie.

References Ackermann, H.-W. (1992), Frequency of morphological phage descriptions. Arch. Viroi., 124, 201-209 Ackermann, H.-W. & DuBow, M.S. (1987a), Viruses of Prokaryotes. Vol. 1. General properties of bacteriophages (pp. 71 and 116-123). CRC Press, Boca Raton, FL. Ackermann, H.-W. & DuBow, M.S. (1987b), Viruses of Prokaryotes. Vol. II. Natural groups of bacteriophages (pp. 85-100 and 121-127). CRC Press, Boca Raton, FL.

Ackermann, H.-W. & Eisenstark, A. (1974), The present state of phage taxonomy. Intervirology, 3, 201-219. Aebi, U., Bijlenga, R.K.L., Ten Heggeler, B., Kistler, J., Steven, A.C. & Smith, P.R. (1976), Comparison of the structural and chemical properties of giant T-even phage heads. J. Supramol. Struct., 5, 475-495. Francki, R.I.B., Fauquet, C.M., Knudson, D.L. & Brown, F. (1991), Classification and nomenclature of viruses. Fifth Report of the International Committee on Taxonomy of Viruses. Arch. Viroi. Suppl. 2, 161-166. Gershman, M. (1976a), A phage typing system for Salmonella binza. Pubi. Hlth Lab., 34, 97-99. Gershman, M. (1976b), A phage typing system for salmonellae: S. senftenberg. J. Milk Food Technol., 39, 682-683. Gershman, M. (1977), Singlephage-typingset for differentiating salmonellae. J. Clin. MicrobioL, 5, 302-314. Gershrnan, M. & Markowsky, G. (1983), Reduced set of phages for typing salmonellae. J. Clin. Microbiol., 17, 240-244. Hohn, T., Hohn, B., Engel, A., Wurtz, M. & Smith, P.R. (1979), Isolation and characterization of the host protein groE involved in bacteriophage ), assembly. J. moi. BioL, 129, 359-373. Kasatiya, S.S. & Nicolle, P. (1978), Phage typing, in "CRC Handbook of Microbiology", 2nd ed. (A.I. Laskin & H.A. Lechevalier)(pp. 699-715). CRC Press, Boca Raton, FL. Luftig, R. (1967), An accurate measurement of the catalase crystal period and its use as an internal marker for electron microscopy. J. Ultrastruct. Res., 20, 91-102. McCorquodale, J.M. & Warner, H.R. (1987), Bacteriophage T5 and related phages, in "The bacteriophages" (R. Calendar) (pp. 439-475). Plenum Press, New York. Werquin, M., Ackermann, H.-W. & Levesque, R.C. (1988), A study of 33 bacteriophages of Rhizobium meliloti. Appl. Environm. Microbiol., 54, 188-196.