phage, 61 nm in diameter, seemed to have a tail-like appendage. All phages .... (3) Phage with empty head and swollen tail tip; this feature may be due to fibers.
JOURNAL OF VIROLOGY, Mar. 1974, p. 706-711 Copyright O 1974 American Society for Microbiology
Vol. 13, No. 3 Printed in U.S.A.
Unusual Bacteriophages in Salmonella newport HANS-W. ACKERMANN, SANDRA PETROW, AND SHANTI S. KASATIYA Department of Microbiology, Faculty of Medecine, Laval University, Quebec 10, and Quebec Department of Social Affairs, Laboratory Service, Ville-de-Laval, P. Q., Canada Received for publication 13 August 1973
Six phages were isolated from sewage and from a lysogenic strain. Three of them, belonging to a new morphological group, had contractile tails and elongated heads with axial ratios of 2.4: 1. Two phages, possessing short tails and very long heads with axial ratios of 3.5: 1, were new isolates of an extremely rare group. Phages of both groups formed polyheads of various sizes and shapes. The last phage, 61 nm in diameter, seemed to have a tail-like appendage. All phages had double-stranded DNA, were active on enterobacteria only, and differed in their host range. The first five phages seemed to be Salmonella specific.
While selecting typing phages for Salmonella newport (S. S. Kasatiya, S. Petrow, and H.-W. Ackermann, manuscript in preparation), we found several phages of very unusual morphology. Some of them seemed to be representatives of a new morphological group. Others resembled the rare bacilliform phages which had been described as early as 1942 (20, 23), then reisolated some 15 years later (6, 13), and subsequently lost (G. Bartsch and V. Bystrick'y, personal communications). -In addition, we found an apparently tailless virus which was considerably larger than 4FX-type phages. These findings motivated us to study these unusual phages in detail. (Presented at the 23rd Annual Meeting of the Canadian Society of Microbiologists, June 1973, Edmonton, Alberta, Canada.) MATERIALS AND METHODS Bacteriophages and bacteria. All phages were isolated by one of us and propagated on Salmonella newport strains. Phages 16-19, 20-36, 20.2, 7-11, and 40.3 were isolated from sewage and grown on strain C487-69. Phage 1412 was produced by strain C1412-72, upon induction by mitomycin C, and grown on strain C178. Methods of phage multiplication, induction, and preparation of specimens for electron microscopy were identical to those described elsewhere (3), except that phages were grown only for 3 h and the lysates were not further purified by density gradient centrifugation. Phage titers were 107 to 108 PFU/ml. Electron microscopy. Phages were stained with 2% potassium phosphotungstate at pH 7.2, or with 2% uranyl acetate at pH 4.5, and studied with a Philips EM 300 electron microscope. Magnification was monitored with T2 phage tails which showed, including the base plate, a length of 120 nm, thus corresponding well with known data (16, 26). 706
Nucleic acid type and host range. The type of nucleic acid was determined according to Bradley (8). The host range was studied by testing undiluted phage suspensions on 150 bacterial strains from our collections (see Table 2).
RESULTS The phages belong to three different morphological groups. Five of them had elongated or isometric capsids and tails which were either contractile or very short. One phage seemed to have no tail at all. Their main dimensions are listed in Table 1. Phages 16-19, 20-36, and 20.2 (Fig. 1-6) formed turbid plaques of about 0.1 mm in diameter and were of identical morphology. Their heads resembled superficially those of T-even phages and appeared to be mostly oval (Fig. 1 and 5), measuring up to 60 nm in diameter. However, when deeply embedded in potassium phosphotungstate or after staining with uranyl acetate, heads showed parallel sides and appeared to be long and narrow (Fig. 2). As T-even phages, they possessed apical and lateral angles (Fig. 1) which have been names a and 3, respectively (7). When measured between opposite lateral angles, the head diameter was 43 nm, thus giving a length-to-width ratio of 2.4:1. Neither edges nor capsomeres were seen. On a few occasions, some malformed heads were found. They were either abnormally long with a tail (Fig. 4) or complex membranous structures of no definite shape (Fig. 6). Tails of these phages were complex and consisted of a neck of 10 x 7 nm, a contractile sheath, a hollow tail tube or "core", a base plate, and at least two tiny fibers. Tails were connected to heads by a double-disk structure,
UNUSUAL PHAGES IN S. NEWPORT
VOL. 13, 1974
TABLE 1. Main dimensions of Salmonella newport phagesa Phage
Determination
Length (nm) Head diameters" (nm) sides (nm) angle a(') angle ,) Tail tube sheath fibers
16-19, 20 36, 20.2
7-11
7-1,
1412
203
178
61
167, 48 136, 29 115.1 122.2
61 31 60
104, 43 77, 26 116 122
707
seemed to be fixed only by a small part of their surface (Fig. 15). After acridine orange staining, all phages showed a green fluorescence which persisted after treatment with tartaric acid. They therefore contained double-stranded DNA. The phages were active on enterobacteria only, differed in their host range, and seemed to be mostly Salmonella specific except for phage 1412, which also lysed Escherichia coli and Shigella flexneri (Table 2).
DISCUSSION Tailed bacteriophages have been divided into 100x7 14x9 9x3c three basic morphological types, A, B, and C. 91 x 14 Type A is comprised of phages with contractile 20 x 2 15 x 2 tails, type B is comprised of phages with long, a Measured on 20 potassium phosphotungstate- noncontractile tails, and type C is comprised of stained full particles. short-tailed phages such as T7 (9). According to 'Transverse diameters of elongated heads were head shape, these types may be further divided measured between two opposite lateral angles; all into subtype 1 for isometric heads, subtype 2 for other were measured between opposite apices. moderately elongated heads such as those of c Apical protrusion. T-even phages, and subtype 3 for very long one disk being inside and the other outside the heads having axial ratios of 2.4 to 3.4 (1). head; only the inner disk is illustrated (Fig. 4). Phages 16-19, 20-36, and 20.2 had contractile The sheath showed no transverse striations. tails and prolate heads with axial ratios of about Upon contraction, it became 37 x 19.6 nm in 2.4:1. They belong therefore to group A3 and size, and the base plate, 19.6 x 2 nm, separated are, to our knowledge, the first representatives of this group described. from the sheath (Fig. 5). Phages 7-11 and 40.3 have outstanding diPhages 7-11 and 40.3 (Fig. 7-12) formed clear plaques of about 0.5 mm in diameter and were mensions and are among the largest bacterial morphologically identical. Their heads were viruses known. They have very short tails and extremely long and, as in the previous phages, even longer heads than the phages of the appeared oval, showing transverse diameters of previous group and belong thus to group C3, up to 65 nm. They too had apical and lateral which is extremely rare (2). These phages are angles and parallel sides (Fig. 8-10). Their real obviously new isolates of the bacilliform colidiameter, as measured between two opposite phages, which are among the first viruses seen lateral angles, was 48 nm, the axial ratio there- in the electron microscope (20, 23). Similar fore being 3.5: 1. Neither edges nor capsomeres coliphages were isolated later with head dimenwere seen, although the head membrane was sions reported as 140 to 160 x 40 to 70 nm (6) or fairly rigid. Tails were very short and seemed to 100 to 120 x 40 to 50 nm (10-12). However, possess one or two short fibers (Fig. 8). These these early micrographs were taken on shadphages also produced regularly sizable numbers owed specimens, did not show the phage tails, of polyheads which were hollow tubes of about or were otherwise unsatisfactory. 55 nm in diameter and ranged from 190 to 1,020 Phages of the A3 and C3 groups have several nm in length (Fig. 11 and 12). These tubes were interesting properties. Both form polyheads of more or less irregular in shape, were closed at various sizes and shapes. A3-group phages proone or both ends, and even possessed tails. duce abnormally long heads and membranous Phage 1412 (Fig. 13-15) formed clear plaques structures which are to be interpreted as a of 0.5 mm in diameter and was produced upon variety of polyheads, since similar structures induction by mitomycin C. Phage 1412 was were found in phages of Bacterium anitratum isometric, rounded, or slightly angular in out- (3) and in phages T7 and Y (4). As for the line (Fig. 13 and 14), had a single capsid C3-group phages, the polyheads are much more without envelope, and apparently had no tail. regular but always empty and penetrated by the However, on some occasions a discrete flat phosphotungstate, therefore suggesting that the protrusion at one apex could be observed (Fig. assembly of normal heads may depend on the 13 and 14). Phages adsorbed to bacterial debris presence of a previously synthetized and closely
708
-a5710
ACKERMANN, PETROW, AND KASATIYA
J. VIROL.
a~~~~~~~~~~~~~~
2
1
5 h;_ .'
1.
I
FIG. 1-6. Phage 16-19; potassium phosphotungstate staining except for Fig. 2; x297,000; bars indicate 100 nm. (1) Normal phage with full head showing the location of angles a and fi, an extended tail, and small tail fibers (arrow). (2) Normal phage after uranyl acetate negative staining. Note the parallel sides and some positive staining of the head. (3) Phage with empty head and swollen tail tip; this feature may be due to fibers folded along the extended sheath. (4) Particle with contracted tail, showing a disk (arrow) inside a head of abnormal size. (5) Phages adsorbed to bacterial debris. Note head flattening and the separation of sheath and base plate. (6) Irregular, more or less spiral-shaped polyhead.
VOL. 13, 1974
709
UNUSUAL PHAGES IN S. NEWPORT
10
14
K
15 FIG. 7-15. Phages 7-11 and 1412; potassium phosphotungstate staining except for Fig. 10 and 14; x297,000; bars indicate 100 nm. Fig. 7-12 are phage 7-11. (7) Normal aspect of intact particles. (8) Empty, flattened phage with fiber-like tail appendages (arrow). (9) Phage deeply embedded in potassium phosphotungstate and (10) particle after uranyl acetate positive staining, both showing narrow heads. (11 and 12) Partially closed polyheads displaying angles. Fig. 13-15 are phage 1412. (13) Two full, rounded particles, one showing a protrusion (arrow). (14) Positively stained phage with apical protrusion (arrow). Note the shrinking of the particle. (15) Phages adsorbed to bacterial debris.
packed nucleic acid core. In addition, it is interesting that in both groups the dimensions of angles a and A, determined with limits of error of 0.8-1.70, are very close to those of T-even type phage heads (116.20 and 123.90, respectively; 3) and to those of B. anitratum phages with elongated heads (116.70 and 123.20, respectively; 3). Moreover, heads of our C3-group phages resemble those of Caulobacter phages oCbK and OCb13 which have axial ratios of 3 to 3.4: 1 and long, noncontractile tails (5, 21, 25). Finally, viable structural aberrations of T-even phages have recently been described which contain DNA and have heads of up to 44
times the normal length (15, 17). These features suggest that at least some of the elongated phage heads are constructed according to the same geometrical principles. Phage 1412 is unusual because it has doublestranded nucleic acid and apparently no tail. Only a few tailless phages of comparable size have been reported, such as a series of Klebsiella phages 45 to 50 nm in diameter which are in part serologically related to tailed Klebsiella phages (18). In our interpretation, the tailless particles could be heads in excess, but the complete phages were not seen. Other tailless phages more than 60 nm in diameter were
710
ACKERMANN, PETROW, AND KASATIYA
J. VIROL
TABLE 2. Host range of Salmonella newport phages No. of strains
Phagea
Enterobacteria Tested
Enterobacter aerogenes Escherichia coli Klebsiella pneumoniae Proteus mirabilis Salmonella enteritidis S. enteritidis S. enteritidis S. newport S. newport S. newport S. newport S. paratyphi B S. thompson S. thompson S. thompson S. typhi S. typhimurium S. typhimurium Serratia marcescens Shigella flexneri Shigella sonnei Total
5 20 10 10 10 10
5 5 5 5 5 5
Sensitive
1
1 2 6 2 2 1 5 2 1 2 1 3 2
105
20.2
7-11
40.3
-
-
-
-
_
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
+
+ +
+ _ _ + + + _ _
+ + +
+ + + + + +
+
+ + +
+
+ + +
+
+
+
_ + +
_ + +
-
+
+
_ _ _ _ _
+
_ _ __ +
-
-
-
-
-
-
1
10
20-36
16-19
-
-
-
-
+
+ -
+
_ + + _ +
_
+ + _
-
-
1412
+
-
_
32
a, lysis; -, no lysis. No phage was active on Bacillus, Bacterium anitratum, Micrococcus, Pseudomonas, or Staphylococcus (10, 10, 5, 15, and 5 strains, respectively).
reported for Bdellovibrio (19), Spirillum (14), and the blue-green algae Synechococcus and Microcystis (24). The Bdellovibrio phage was described as having single-stranded DNA and a double capsid, whereas the algal virus was later found to have a short tail (22). It would be surprising to find a relatively large, tailless virus with double-stranded DNA in enterobacteria. We therefore interpret the apical protrusion of phage 1412 as a tail equivalent and consider this phage a member of the Cl group. ACKNOWLEDGMENTS This investigation was supported by grants 604-7-804 (H.-W. A) and 604-7-833 (S. S. K.) from the Canadian Department of National Health and Welfare. We are indebted to Marie-Christine Montegu for able technical assistance. LITERATURE CITED 1. Ackermann, H.-W. 1969. Bacteriophages-proprietes et premieres etapes d'une classification. Pathol. Biol.
17:1003-1024. 2. Ackermann, H.-W. 1973. Tailed bacteriophages: listing by morphological groups, p. 579-607. In A. I. Laskin and H. A. Lechevalier (ed.), Handbook of microbiology, vol. 1. Chemical Rubber Co. Press, Cleveland, Ohio. 3. Ackermann, H.-W., G. Brochu, and G. Cherchel. 1973.
4.
5. 6. 7.
8.
9. 10.
Structure de trois phages de Bacterium anitratum (groupe (B5W)). J. Microsc. (Paris) 16:215-224. Ackermann, H.-W., and F. Poty. 1973. Polytetes chez les phages T7 et Y. Zentralbl. Bakteriol. Parasitenk. Infektionskr. Hyg. Abt. Orig. 223:421-425. Agabian-Keshishian, N., and L. Shapiro. 1970. Stalked bacteria: properties of deoxyribonucleic acid bacteriophage 0CbK. J. Virol. 5:795-800. Bartsch, G. 1960. Ein selten beobachteter Coliphage. Jenaer Rundschau 5:7-10. Bradley, D. E. 1965. The structure of the head, collar and base-plate of "T-even" type bacteriophages. J. Gen. Microbiol. 38:395-408. Bradley, D. E. 1966. The fluorescent staining of bacteriophage nucleic acids. J. Gen. Microbiol. 44:383-391. Bradley, D. E. 1967. Ultrastructure of bacteriophages and bacteriocins. Bacteriol. Rev. 31:230-314. Bystricky, V. 1962. Negative staining of a rod-shaped bacteriophage. J. Electronmicrosc. (Tokyo)
11:125-127. 11. Bystricky, V. 1964. On the structure of some bacteriophages, p. 557-558. In M. Titlbach (ed.), Proceedings of the 3rd European regional conference on electron microscopy, vol. B. Publishing House of the Czechoslovak Academy of Sciences, Prague. 12. Bystricky, V., V. Drahos, M. Mulczyk, A. Przondo-Hessek, and S. Slopek. 1964. On the structure of some bacteriophages. Acta Virol. 8:369-372. 13. Bystricky, V., and L. Sevcovicova. 1958. Morphologische und einige serologische Eigenschaften eines stabchenformigen Coli-Bakteriophagen. Zentralbl. Bakteriol. Parasitenk. Infektionskr. Hyg. Abt. Orig. 171:25-44. 14. Clark-Walter, G. D., and S. B. Primrose. 1971. Isolation
VOL. 13, 1974
15.
16. 17.
18.
19.
20.
UNUSUAL PHAGES IN S. NEWPORT
and characterization of a bacteriophage SiI for Spirillum itersonii. J. Gen. Virol. 11:139-145. Cummings, D. J., V. A. Chapman, S. S. DeLong, and N. L. Couse. 1973. Structural aberrations in T-even bacteriophage. HI. Induction of "lollipops" and their partial characterization. Virology 54:245-261. Cummings, D. J., and L. M. Kozloff. 1960. Biophysical properties of bacteriophage T2. Biochim. Biophys. Acta 44:445-458. Doermann, A. H., F. A. Eiserling, and L. Boehner. 1973. Genetic control of capsid length in bacteriophage T4. I. Isolation and preliminary description of four new mutants. J. Virol. 12:374-385. Gabrilovich, I. M., S. I. Lukelyeva, W. S. Polupanov, and S. B. Stefanov. 1968. Molecular structure of Klebsiella phages and their specificity. Arch. Immunol. Ther. Exp. 16:870-880. Hashimoto, T., D. L. Diedrich, and S. F. Conti. 1970. Isolation of a bacteriophage for Bdellovibrio bacteriovorus. J. Virol. 5:97-98. Kottmann, U. 1942. Morphologische Befunde aus taches
21.
22. 23. 24.
25.
26.
711
vierges von Colikulturen. Arch. Gesamte Virusforsch. 2:388-396. Leonard, K. R., A. K. Kleinschmidt, N. Agabian-Keshishian, L. Shapiro, and J. V. Maizel. 1972. Structural studies on the capsid of Caulobacter crescentus bacteriophage OCbK. J. Mol. Biol. 71:201-216. MacKenzie, J. J., and R. Haselkorn. 1972. Physical properties of blue-green algal virus SM-1 and its DNA. Virology 49:497-504. Ruska, H. 1942. Morphologische Befunde bei der bakteriophagen Lyse. Arch. Gesamte Virusforsch. 2:345-387. Safferman, R. S., I. R. Schneider, R. L. Steere, M. E. Morris, and T. 0. Diener. 1969. Phycovirus SM-1: a virus infecting unicellular blue-green algae. Virology 37:386-395. Schmidt, J. M., and R. Y. Stanier. 1965. Isolation and characterization of bacteriophages active against stalked bacteria. J. Gen. Microbiol. 39:95-107. Tikhonenko, A. S. 1968. Ultrastructure of bacterial viruses. Izdadelstvo "Nauka", Moscow and Plenum Press, New York and London (1970).
