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Morphology of Listeria innocua bacteriophages
Ann. Virol. (Inst. Pasteur) 1983, 134 E, 245-250
MORPHOLOGY OF L I S T E R I A
INNOCUA
BACTERIOPHAGES
by J. Rocourt Q), H. W. Ackermann (0, M. Martin (2), A. Schrettenbrunner (3) and H. P. R. Seeliger (0
(1) Unitd d'l~cologie Bact~rienne (Pr H. H. Mollaret), Inslilut Pasteur, 75724 Paris Cedex 15, (0 D~parlement de Microbiologie, Facultd de Mddecine, Universitd Laval, Quebec, P. Q. G1K 7P4 (Canada) and (3) Institut ]i~r Hygiene und Mikrobiotogie, Universittit Wi~rzburg, 8700 Wi~rzburg (FRG)
SUMMARY Seven temperate bacteriophages of Listeria innocua were studied by electron microscopy. Two phages had contractile tails and belonged to the Mgoviridae family. Up to now, viable phages of this type had not been reported in the genus Listeria. Five phages with long, non-contractile tails belonged to the Stgloviridae family and represented two known species of Listeria phages. KEY-WORDS: Bacteriophage, Ultrastructure, Listeria innocua; Mgoviridae, Slgloviridae.
INTRODUCTION All Listeria phages known thus far had been isolated from lysogenic Lisleria monocgtogenes [1, 4, 5, 14]. Recently, one of us proposed a new species, Lisleria innocua, which includes non-haemolytic and apathogenic strains of L. monocgtogenes belonging to serovars 6a, 6b and various undesignated serovars [12, 13J. This study describes the morphology of seven bacteriophages isolated from non-induced strains of serovars 6a and 6b [ 10], which correspond phenotypically to the L. innocua genomic group [9, 11].
M&TERIALS AND METHODS Seven bacteriophages were studied (table I). The origin, methods of propagation and host ranges of six of these phages have been published elsewhere [10]. A new M a n u s c r i t re~u le 9 f6vrier 1983, accept6 le 21 m a r s 1983.
246
J. ROCOURT AND COLL.
phage called 184 was isolated from broth culture of a strain of serovar 6a and purified in the same manner as the other phages [10]. Phages were sedimented at 70,000 g for 90 rain in an ,~ International B-60 ~ ultracentrifuge using an SB-405 rotor, and washed twice with 0.1 M ammonium acetate (pH 7.0). Pellets were deposited on carbon-coated Formvar grids, stained with 2 % potassium phosphotungstate (PT) (pH 7.2) or 2 ~o uranyl acetate (UA) (pH 4.0), and studied in a <(Philips EM 300 ,~electron microscope. Magnification was monitored with catalase crystals (Polaron Equipment Ltd., London) [6].
R E S U L T S AND DISCUSSION The seven phages studied had isometric heads and either contractile or long, non-contractile tails. They represented three morphological types, illustrated in figures 1-7. Their main dimensions are listed in table I. Heads were icosahedral, as evidenced by the simultaneous occurrence of heads with pentagonal and hexagonal outlines (fig. 4 and 5). Two phages (4211 and 4286) had contractile tails and belonged to the Mgoviridae family [7]. Tails consisted of a neck of 10 • 8 rim, a sheath with transverse striations of about 3 nm periodicity and an 8 nm-wide core. Contracted sheaths measured 140 • 20 n m and were frequently seen in variable positions on the tail core, suggesting t h a t t h e y had become detached (fig. 2). The phages lysed both L. innocua and L. monocgtogeues serovars 4b and 5 [10]. These phages are something of a rarity. Among at least 255 phages of Gram-positive asporogenous rods, only 20 have contractile tails. Fifteen are Lactobacillus phages, while none had previously been found in Listeria [8].
PT ~ phosphotungstate. UA = uranyl acetate.
FIG. 1-7. - -
Various L. i n n o c u a bacteriophage species.
A l l p h a g e s a r e n e g a t i v e l y s t a i n e d u n l e s s o t h e r w i s e i n d i c a t e d . F i g u r e s 1-4 a n d 6-7: U A ; f i g u r e 5: PT. (X 297,000). Bars represent 100 nm. FIG. 1 - 5 . - - S p e c i e s 4 2 1 1 , p h a g e 4 2 8 6 . 1 a n d 2. - - P h a g e w i t h e x t e n d e d o r c o n t r a c t e d
tail (UA).
3. - - A f t e r U A p o s i t i v e s t a i n i n g ; n o t e s h r u n k e n , h e a v i l y - s t a i n e d h e a d . 4 a n d 5. - - P e n t a g o n a l h e a d s a f t e r U A a n d P T s t a i n i n g , r e s p e c t i v e l y . FIG. 6. - - S p e c i e s 2 6 7 1 , p h a g e 5 3 3 7 s h o w i n g b a s e - p l a t e o r i e n t e d u p w a r d s ( U A ) . F r o . 7. - - S p e c i e s 2 6 8 5 , p h a g e 4 2 9 2 .
Fro. I h 7
Ann. Virol. (Inst. Pasteur.) 134 E,
n ~ 2, 1983.
17
MORPHOLOGY OF L I S T E R I A BACTERIOPHAGES TABLE I.
-
-
249
Main dimensions of ~ Listeria innocua ~ bacteriophages.
Phage
Dimensions (nm)
Nb of particles measured
Stain
Head
Tail
Family
Species
Myoviridae
4211
4211,4286
20 20
PT UA
59 62
232 • 14 230 • 18
Styloviridae
26~1
184,4276,4277,5337
2685
4292
20 20 5 5
PT UA PT UA
58 57 57 59
296 294 253 249
• • • •
6 9 6 9
UA ~ u r a n y l acetate; P T ~ p h o s p h o t u n g s t a t e . Head diameters were m e a s u r e d between opposite apices.
All other 47 known Listeria phages have non-contractile tails [1, 4, 5, 14]. According to Bradley and Dewar [3], L. monocgtogenes (serovar not reported) produced phage-like bacteriocidal particles called (( monocins , with contractile tails 240 nm in length. These particles differed from our phages in various properties: 1) most of t h e m had no heads, but did have conspicuous, six-pointed base-plates; 2) heads, if present, were always e m p t y and v e r y large, measuring 110 n m in diameter [3]. It was concluded t h a t our phages represented a new species which we called c( 4211 )~ after one of its m e m b e r phages. Five phages had long, non-contractile tails, thus belonging to the Stgloviridae family [7]. According to the tail length, t h e y represented two phage species already observed in L. monocgtogenes, t h a t is, species 2671 (phages 184, 4276, 4277 and 5337) and species 2685 (phage 4292). Their host range included both L. innocua and L. monocytogenes serovar 5 [10]. Tails were more or less flexible, showed transverse striations of about 5 n m periodicity and t e r m i n a t e d in a base plate in the form of a six-pointed star, as described earlier [1]. Preparations of phages belonging to species 2671 sometimes contained abnormally long or short tails with or without e m p t y heads (not shown). Species 2671 has already been observed in a L. innocua strain formerly attributed to L. monocytogenes (phage 1090) [1, 2]. It can only be conjectured as to whether t h e Listeria phages of Chiron et al. [4] and Sword and Pickett [14] include phages of L. innocua, since these authors did not indicate the serovars of their strains.
RESUMg MORPHOLOGIE
DE
BACTI~RIOPHAGES DE
INNOCUA ~
Sept bact6riophages temp~r~s de Listeria innocua ont ~t~ ~tudi~s au microscope ~lectronique. D e u x phages pr~sentent une queue contractile
250
J. ROCOURT AND COLL.
et a p p a r t i e n n e n t h la famille des Mgoviridae. De tels phages viables n ' o n t pas encore ~t6 d~crits pour le genre Listeria. Cinq phages poss~dent une queue non contractile et a p p a r t i e n n e n t h la famille des Stgloviridae. Ils repr6sentent deux esp~ces d~jh connues de phages de Listeria. MOTS-CLI~S : Bacteriophage, Ultrastructure, Listeria innocua; Mgoviridae, Stgloviridae.
ACKNO~u J. Rocourt received a travel grant from the French (( Minist6re des Affaires ]~trang~res )) and from the (( Minist6re des Affaires Intergouvernementales de la Province de Qu6bec )) (Canada).
REFERENCES
[1] ACKERMANN,H.-W., AUDURIER,A. & ROCOURT, J., Morphologie de bactdrio[21 [3] [4] [5] [6]
[7] [8] [9] [10] [11]
[12] [13]
[14]
phages de Listeria monocytogenes. Ann. Virol. (Inst. Pasteur), 1981, 132 E, 371-382. AUDURIER,A., ROCOURT, J. & COURTIEU, A.-L., Isolement et caractdrisation de bact6riophages de Lisleria monocgtogenes. Ann. Microbiol. (Inst. Pasteur), 1977, 128 A, 185-198. BRADLEY, D. E. & DEWAR, C.-A., The structure of phage-like objects associated with non-induced bacteriocinogenic bacteria. J. gen. Microbiol., 1966, 45, 399-408. CHmON, J. P., MAURAS, P. & DENIS, F., Ultrastructure des bactdriophages de Listeria monocgtogenes. C. R. Soc. Biol. (Paris), 1977, 171, 488-492. JASINSKA, S., Bacteriophages of lysogenic strains of Listeria monocgtogenes. Acta microbiol, pol., 1964, 13, 29-44. LUFTm, R., An accurate measurement of the catalase crystal period and its use as an internal marker for electron microscopy. J. Ultrastruct. Res., 1967, 20, 91-102. MATTHEWS,R. E. F., Classificatio~a and nomenclature of viruses. Intervirology, 1982, 17, 1-199. REANNEY, D. C. & ACKERMANN,H.-W., Comparative biology and evolution of bacteriophages. Advanc. Virus Res., 1982, 27, 205-280. ROCOURT, J., GRIMONT, F., GRIMONT, P. A. D. & SEELIGER, H. P. R., DNA relatedness among serovars of Listeria monocgtogenes sensu lalo. Curr. Microbiol., 1982, 7, 383-388. ROCOURT, J., SCHRETTENBRUNNEH,i . & SEELIGER, H. P. l~., Isolation of bacteriophages from Lisleria innocua and Listeria monocglogenes serovar 5. ZbI. Bakt., I. Abt. Orig., 1982, 251, 505-511. RocOUnT, J., SCH1RETTENBRUNNER,A. & SEELIGER, H, P. R., Diffdrenciation biochimique des groupes gdnomiques de Listeria monocgtogenes (sensu taro). Ann. Microbiol. (Inst. Pasteur), 1983, 134 A, 65-71. SEELIGER, H. P. R., Apathogene Listerien : Listeria innocua sp. n. (Seeliger & Schoofs, 1977). Zbl. Bakt., I. Abt. Orig., 1981, 249, 487-493. SEELIGER, H. P. R. & HiJHNE, K., Serotyping of Listeria monocytogenes and related species, in (( Methods in microbiology )) (T. Bergan & J. R. Norris) (pp. 31-49), 13. Academic Press, New York, Toronto, Sydney, San Francisco, 1979. SWORD, C. P. & PICKETT, M. J., The isolation and characterization of bacteriophages from Listeria monocytogenes. J. gen. Microbiol., 1961, 25, 241248.
MORPHOLOGY OF L I S T E R I A
INNOCUA
BACTERIOPHAGES
by J. Rocourt Q), H. W. Ackermann (0, M. Martin (2), A. Schrettenbrunner (3) and H. P. R. Seeliger (0
(1) Unitd d'l~cologie Bact~rienne (Pr H. H. Mollaret), Inslilut Pasteur, 75724 Paris Cedex 15, (0 D~parlement de Microbiologie, Facultd de Mddecine, Universitd Laval, Quebec, P. Q. G1K 7P4 (Canada) and (3) Institut ]i~r Hygiene und Mikrobiotogie, Universittit Wi~rzburg, 8700 Wi~rzburg (FRG)
SUMMARY Seven temperate bacteriophages of Listeria innocua were studied by electron microscopy. Two phages had contractile tails and belonged to the Mgoviridae family. Up to now, viable phages of this type had not been reported in the genus Listeria. Five phages with long, non-contractile tails belonged to the Stgloviridae family and represented two known species of Listeria phages. KEY-WORDS: Bacteriophage, Ultrastructure, Listeria innocua; Mgoviridae, Slgloviridae.
INTRODUCTION All Listeria phages known thus far had been isolated from lysogenic Lisleria monocgtogenes [1, 4, 5, 14]. Recently, one of us proposed a new species, Lisleria innocua, which includes non-haemolytic and apathogenic strains of L. monocgtogenes belonging to serovars 6a, 6b and various undesignated serovars [12, 13J. This study describes the morphology of seven bacteriophages isolated from non-induced strains of serovars 6a and 6b [ 10], which correspond phenotypically to the L. innocua genomic group [9, 11].
M&TERIALS AND METHODS Seven bacteriophages were studied (table I). The origin, methods of propagation and host ranges of six of these phages have been published elsewhere [10]. A new M a n u s c r i t re~u le 9 f6vrier 1983, accept6 le 21 m a r s 1983.
246
J. ROCOURT AND COLL.
phage called 184 was isolated from broth culture of a strain of serovar 6a and purified in the same manner as the other phages [10]. Phages were sedimented at 70,000 g for 90 rain in an ,~ International B-60 ~ ultracentrifuge using an SB-405 rotor, and washed twice with 0.1 M ammonium acetate (pH 7.0). Pellets were deposited on carbon-coated Formvar grids, stained with 2 % potassium phosphotungstate (PT) (pH 7.2) or 2 ~o uranyl acetate (UA) (pH 4.0), and studied in a <(Philips EM 300 ,~electron microscope. Magnification was monitored with catalase crystals (Polaron Equipment Ltd., London) [6].
R E S U L T S AND DISCUSSION The seven phages studied had isometric heads and either contractile or long, non-contractile tails. They represented three morphological types, illustrated in figures 1-7. Their main dimensions are listed in table I. Heads were icosahedral, as evidenced by the simultaneous occurrence of heads with pentagonal and hexagonal outlines (fig. 4 and 5). Two phages (4211 and 4286) had contractile tails and belonged to the Mgoviridae family [7]. Tails consisted of a neck of 10 • 8 rim, a sheath with transverse striations of about 3 nm periodicity and an 8 nm-wide core. Contracted sheaths measured 140 • 20 n m and were frequently seen in variable positions on the tail core, suggesting t h a t t h e y had become detached (fig. 2). The phages lysed both L. innocua and L. monocgtogeues serovars 4b and 5 [10]. These phages are something of a rarity. Among at least 255 phages of Gram-positive asporogenous rods, only 20 have contractile tails. Fifteen are Lactobacillus phages, while none had previously been found in Listeria [8].
PT ~ phosphotungstate. UA = uranyl acetate.
FIG. 1-7. - -
Various L. i n n o c u a bacteriophage species.
A l l p h a g e s a r e n e g a t i v e l y s t a i n e d u n l e s s o t h e r w i s e i n d i c a t e d . F i g u r e s 1-4 a n d 6-7: U A ; f i g u r e 5: PT. (X 297,000). Bars represent 100 nm. FIG. 1 - 5 . - - S p e c i e s 4 2 1 1 , p h a g e 4 2 8 6 . 1 a n d 2. - - P h a g e w i t h e x t e n d e d o r c o n t r a c t e d
tail (UA).
3. - - A f t e r U A p o s i t i v e s t a i n i n g ; n o t e s h r u n k e n , h e a v i l y - s t a i n e d h e a d . 4 a n d 5. - - P e n t a g o n a l h e a d s a f t e r U A a n d P T s t a i n i n g , r e s p e c t i v e l y . FIG. 6. - - S p e c i e s 2 6 7 1 , p h a g e 5 3 3 7 s h o w i n g b a s e - p l a t e o r i e n t e d u p w a r d s ( U A ) . F r o . 7. - - S p e c i e s 2 6 8 5 , p h a g e 4 2 9 2 .
Fro. I h 7
Ann. Virol. (Inst. Pasteur.) 134 E,
n ~ 2, 1983.
17
MORPHOLOGY OF L I S T E R I A BACTERIOPHAGES TABLE I.
-
-
249
Main dimensions of ~ Listeria innocua ~ bacteriophages.
Phage
Dimensions (nm)
Nb of particles measured
Stain
Head
Tail
Family
Species
Myoviridae
4211
4211,4286
20 20
PT UA
59 62
232 • 14 230 • 18
Styloviridae
26~1
184,4276,4277,5337
2685
4292
20 20 5 5
PT UA PT UA
58 57 57 59
296 294 253 249
• • • •
6 9 6 9
UA ~ u r a n y l acetate; P T ~ p h o s p h o t u n g s t a t e . Head diameters were m e a s u r e d between opposite apices.
All other 47 known Listeria phages have non-contractile tails [1, 4, 5, 14]. According to Bradley and Dewar [3], L. monocgtogenes (serovar not reported) produced phage-like bacteriocidal particles called (( monocins , with contractile tails 240 nm in length. These particles differed from our phages in various properties: 1) most of t h e m had no heads, but did have conspicuous, six-pointed base-plates; 2) heads, if present, were always e m p t y and v e r y large, measuring 110 n m in diameter [3]. It was concluded t h a t our phages represented a new species which we called c( 4211 )~ after one of its m e m b e r phages. Five phages had long, non-contractile tails, thus belonging to the Stgloviridae family [7]. According to the tail length, t h e y represented two phage species already observed in L. monocgtogenes, t h a t is, species 2671 (phages 184, 4276, 4277 and 5337) and species 2685 (phage 4292). Their host range included both L. innocua and L. monocytogenes serovar 5 [10]. Tails were more or less flexible, showed transverse striations of about 5 n m periodicity and t e r m i n a t e d in a base plate in the form of a six-pointed star, as described earlier [1]. Preparations of phages belonging to species 2671 sometimes contained abnormally long or short tails with or without e m p t y heads (not shown). Species 2671 has already been observed in a L. innocua strain formerly attributed to L. monocytogenes (phage 1090) [1, 2]. It can only be conjectured as to whether t h e Listeria phages of Chiron et al. [4] and Sword and Pickett [14] include phages of L. innocua, since these authors did not indicate the serovars of their strains.
RESUMg MORPHOLOGIE
DE
BACTI~RIOPHAGES DE
INNOCUA ~
Sept bact6riophages temp~r~s de Listeria innocua ont ~t~ ~tudi~s au microscope ~lectronique. D e u x phages pr~sentent une queue contractile
250
J. ROCOURT AND COLL.
et a p p a r t i e n n e n t h la famille des Mgoviridae. De tels phages viables n ' o n t pas encore ~t6 d~crits pour le genre Listeria. Cinq phages poss~dent une queue non contractile et a p p a r t i e n n e n t h la famille des Stgloviridae. Ils repr6sentent deux esp~ces d~jh connues de phages de Listeria. MOTS-CLI~S : Bacteriophage, Ultrastructure, Listeria innocua; Mgoviridae, Stgloviridae.
ACKNO~u J. Rocourt received a travel grant from the French (( Minist6re des Affaires ]~trang~res )) and from the (( Minist6re des Affaires Intergouvernementales de la Province de Qu6bec )) (Canada).
REFERENCES
[1] ACKERMANN,H.-W., AUDURIER,A. & ROCOURT, J., Morphologie de bactdrio[21 [3] [4] [5] [6]
[7] [8] [9] [10] [11]
[12] [13]
[14]
phages de Listeria monocytogenes. Ann. Virol. (Inst. Pasteur), 1981, 132 E, 371-382. AUDURIER,A., ROCOURT, J. & COURTIEU, A.-L., Isolement et caractdrisation de bact6riophages de Lisleria monocgtogenes. Ann. Microbiol. (Inst. Pasteur), 1977, 128 A, 185-198. BRADLEY, D. E. & DEWAR, C.-A., The structure of phage-like objects associated with non-induced bacteriocinogenic bacteria. J. gen. Microbiol., 1966, 45, 399-408. CHmON, J. P., MAURAS, P. & DENIS, F., Ultrastructure des bactdriophages de Listeria monocgtogenes. C. R. Soc. Biol. (Paris), 1977, 171, 488-492. JASINSKA, S., Bacteriophages of lysogenic strains of Listeria monocgtogenes. Acta microbiol, pol., 1964, 13, 29-44. LUFTm, R., An accurate measurement of the catalase crystal period and its use as an internal marker for electron microscopy. J. Ultrastruct. Res., 1967, 20, 91-102. MATTHEWS,R. E. F., Classificatio~a and nomenclature of viruses. Intervirology, 1982, 17, 1-199. REANNEY, D. C. & ACKERMANN,H.-W., Comparative biology and evolution of bacteriophages. Advanc. Virus Res., 1982, 27, 205-280. ROCOURT, J., GRIMONT, F., GRIMONT, P. A. D. & SEELIGER, H. P. R., DNA relatedness among serovars of Listeria monocgtogenes sensu lalo. Curr. Microbiol., 1982, 7, 383-388. ROCOURT, J., SCHRETTENBRUNNEH,i . & SEELIGER, H. P. l~., Isolation of bacteriophages from Lisleria innocua and Listeria monocglogenes serovar 5. ZbI. Bakt., I. Abt. Orig., 1982, 251, 505-511. RocOUnT, J., SCH1RETTENBRUNNER,A. & SEELIGER, H, P. R., Diffdrenciation biochimique des groupes gdnomiques de Listeria monocgtogenes (sensu taro). Ann. Microbiol. (Inst. Pasteur), 1983, 134 A, 65-71. SEELIGER, H. P. R., Apathogene Listerien : Listeria innocua sp. n. (Seeliger & Schoofs, 1977). Zbl. Bakt., I. Abt. Orig., 1981, 249, 487-493. SEELIGER, H. P. R. & HiJHNE, K., Serotyping of Listeria monocytogenes and related species, in (( Methods in microbiology )) (T. Bergan & J. R. Norris) (pp. 31-49), 13. Academic Press, New York, Toronto, Sydney, San Francisco, 1979. SWORD, C. P. & PICKETT, M. J., The isolation and characterization of bacteriophages from Listeria monocytogenes. J. gen. Microbiol., 1961, 25, 241248.