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The fine structure of a pyocin
J. Mol. Biol. (W65) 13,428-431
The Fine Structure of a Pyocin SHIN-ICHI ISHII
Department of Biophysics and Biochemistry Faculty of Science, University of Tokyo, Tokyo
y OSHIMI
NISHI
Scientfiic Instruments Department, Naka Works, Hitachi, Ltd., Katsuta, Japan AND FUJIO EGAMI
Department of Biophysics and Biochemistry Faculty of Science, University of Tokyo, Tokyo, Japan (Received 17 May 1965) Negatively stained specimens of pyocin, a bacteriocin produced by strain R Pseudomonas aeruginosa, were examined by electron microscopy, and a close resemblance of the pyocin to T -even bacteriophage tails was demonstrated with regard to their fine structure. The main structure of the pyocin was coneluded to be a double hollow cylinder, 1200 A long and 150 A in outer diameter, which consisted of a "sheath" and a "core". The sheath appeared capable of contraction. The presence of a "base plate" and "tail fibres" was also suggested. The sheath and the core were found to show markedly different behaviour under various chemical treatments, including those by urea, sodium dodecyl sulphate, acid and alkali. They may be composed of protein subunits which differ from each other in chemical properties.
1. Introduction Jacob (1954) was the first to describe pyocin, an antibacterial substance which was produced by certain strains of Pseudomonas aeruginosa and exerted a specific bactericidal activity typical of bacteriocins. Kageyama & Egami (1962) and Kageyama (1964) have recently succeeded in purifying a pyocin as a homogeneous protein of high molecular weight, from the lysate of ultraviolet- or mitomycin C-induced cells of Ps. aeruqinosa strain R. The purified preparation of pyocin, which was shadowed with chromium, appeared in the electron microscope as rod-like particles with a uniform size. They thought the rods resembled bacteriophage tails. In the present study, the use of the negative staining technique (Brenner & Horne, 1959) led to the elucidation of the fine structure of the pyocin. The presence of at least two structural components, "sheath" and "core", was demonstrated, as in the tails of T-even phages. The behaviour of these structural components under various chemical treatments has also been examined.
2. Materials and Methods The purified pyocin was prepared from the lysate of mitomycin C-induced cells of Ps, aeruginosa strain R by the procedure of Kageyama (1964). Either directly or after various chemical treatments, the pyocin in 0·1 M-NaCl containing 0·005 M-tris buffer (pH 7-2),
428
PLATE
1. An electron micrograph of purified pyocin ( X 250,000).
[facing p. 430
PLATE II. (a) A micrograph at a higher magnification of the same specimen shown in Plate I (X 400,000). (b) Pyocin treated with mild alkali (pH 10,5). Three sets of striations (in directions indicated with arrows) are visible on the surface of an extended sheath (x 400,000). (c) A micrograph at a higher magnification of the same specimen shown in Plate VIII. The contracted sheath appears to accompany a base plate and tail fibres (x 400,000). (d) A micrograph at a higher magnification of the same specimen shown in Plate IV ( X 400,000).
PLATE III. A micrograph of pyocin treated with 0·1 % sodium dodecyl sulphate showing the formation of stretched helical structure from ends of partially disintegrated sheaths. Cores seem to be decomposed completely ( X 250,000).
loooA PLATE IV. Pyocin treated with 6 llil-urea. A number of sheaths or their segments can be seen standing upright, revealing the "cog wheel" arrangement of the subunits ( X 250.000).
PLATE V. Pyocin treated with acid (pH 2,4). The whole of the contracted sheath structure seems to be preserved, while complete disappearance of the core structure occurs also in this case ( X 250,000).
PLATE VI. Pyocin treated under slightly acidic conditions (pH 6). Many small rings may correspond to the sections of disrupted cores seen end-on ( X 250,000).
PLATE VII. Structures resulting from the treatment of pyocin with alkali (pH U·O). These consist of contracted sheaths with or without cores, and detached cores. The sheaths demonstrate disrupted figures frequently (x 250,000).
PLATE VIII. A whole pellet preparation from the induced-cell lysate. Almost all the particles are interpretable as those of pyocin, except a phage-like particle observed in the middle of the field ( X 250,000).
FINE STRUCTURE OF A PYOCIN
429
was diluted five- to tenfold, usually with distilled water, to afford the sample solution of appropriate concentration (0,1 to 0·2 mg proteinjml.) for negative staining. A crude pellet preparation of the lysate was obtained by the following process. The mitomycin O-induced cell lysate (35 ml.) was first treated with I /kg/ml. deoxyribonuclease (Worthington) for 60 min at 37°0 and was cleared of bacterial debris by low-speed centrifugation. The supernatant fraction was centrifuged in the type 40P Hitachi ultracentrifuge for 45 min at 25,000 rev Jrou: (about 60,000 g). The pellet was suspended in 5 ml. of 0·05 M-tris buffer (pH 7,7). and used for the electron microscope investigation. Negatively stained specimens were prepared by depositing a drop of the sample solutions on a micro-grid (Sakata, 1958) and adding another drop of a 2% solution of phosphotungstic acid adjusted to pH 7·0 with potassium hydroxide. The excess solution was withdrawn by touching the edge of the grid with filter paper. The grids were always mounted in the electron microscope with the specimen side away from the electron source, and photographic enlargements were made from the electron microscope plates by inserting them in the enlarger with the emulsion side away from the light source. The type HU-IIA Hitachi electron microscope was used at a magnification of 50,000.
3. Results Plate I is an electron micrograph of the purified pyocin. There are rod-like particles, 1200 A long and 150 A wide, which show striations running nearly at right angles to the particle axes. Their appearance is very similar to that of the T-even bacteriophage tails (Brenner et al., 1959). In addition, particles with "contracted sheaths" are observed in the Plate. "Cores" are missing in some of the particles of this type. The profiles of these contracted sheaths can be interpreted as hollow cylinders 460 A long and 180 A in outer diameter. The core also shows a hollow tubular structure 1200 A long and 57 A in outer diameter. The micrograph Plate II(a) indicates two typical pyocin particles with the extended sheaths at a higher enlargement, where approximately 35 regular striations can be counted along their lengths. Plate II(b) is a selected area of an electron micrograph of the pyocin treated under mild alkaline conditions (pH 10,5) for five hours. In the middle of this Plate there is a representative of the pyocin particles which still have extended sheaths after this treatment. There are two more sets of striations clearly visible at opposite slanting angles (indicated by arrows A and B) on the surface of this particle, besides the cross-striations (arrow C) mentioned above. The fine round particles detectable along these three families of striations may eventually be correlated with the protein subunits which constitute the whole sheath. The "base plate" structure is also visible at a distal end of the pyocin particle in Plate II(b). The contracted sheath in Plate II(c) appears to have pulled up the base plate leaving an end of the core free, and the base plate seems to accompany "tail fibres" . Plate III demonstrates characteristic structures produced by treating pyocin with 0·1 % sodium dodecyl sulphate at pH 7 for about 20 hours. Contracted sheaths have been partly disintegrated, releasing stretched helices from their ends, while all cores have disappeared. The appearance of the stretched helices suggests that a dominant helical array of the sheath subunits is present along a set of the cross-striations observed on the extended sheath. Treatment with 6 M-urea also decomposes the core structures completely and leaves empty contracted sheaths as seen in Plate IV. Partial disruption of the sheaths occurs, but no stretching of the helical array is observed in this case. There are several sheaths or their disrupted segments standing upright. They display the "cog wheel" appearance similar to that described by
430
S. ISHII, Y. NISHI AND F. EGAMI
Brenner et at. (1959) about the contracted sheaths of T2 phage tails. Their enlarged images are shown in Plate II(d). In the case of acid treatment (0-05 N-acetic + 0·05 N-formic acid (pH 2-4), 20 hours), the entire contracted sheath structure appears to be preserved as shown in Plate V, while cores are destroyed completely again. Plate VI, a micrograph of the \ pyocin treated under a slightly acidic condition (pH 6, about 20 hours), exhibits many small rings which may correspond to the sections of disrupted cores seen end-on. A double-ring (indicated with an arrow in Plate VI) is thought to be an end-on view of the contracted sheath still accommodating a piece of the disrupted core. Plate VII shows that alkali-treatment (0-05 M-Na2C03 (pH 11-0), 20 hours) of pyocin produced a variety of altered particles. All the sheaths have been contracted and some detached from cores. In contrast with the cores, the sheaths demonstrate disrupted figures quite frequently. Many sheaths, probably disrupted, are observed end-on, showing a cog wheel appearance again.
4. Discussion Evidence is presented to demonstrate morphological resemblance of pyocin produced by Ps. aeruginosa strain R to T-even phage tails. The main structure of the pyocin is concluded to be a double hollow cylinder which consists of a sheath and a core. The sheath, which seems to be capable of contraction, appears to be composed of numerous protein subunits arranged in screw symmetry (see Plate II(a), (b). It can further be argued that a fundamental helical array of the sheath subunits along the cross-striations (arrow C in Plate II(b» has produced the stretched spirals seen in the specimen treated with 0-1 % sodium dodecyl sulphate (Plate III). The number of the cross-striations is about 35 and these may represent the number of turns of the helix in the extended sheath. On the other hand, about 12 "cogs" can be seen in end-on view of contracted sheaths (Plate II(d». This is not likely to be the number of subunits in one turn of the helix in the extended structure, however, because the increase in diameter of the sheath at the time of contraction suggests that this process accompanies an increase in the number of subunits per turn, as already discussed by Brenner et at. (1959). Chemical studies on purified contracted sheaths, now in progress, are expected to lead to more precise information on all the subunits in the sheath. The structure of cores in pyocin particles is found to be very labile upon treatment with urea (6 M) or sodium dodecyl sulphate (0-1 %). Complete disappearance of the core structure in electron micrographs was seen even with the specimen treated with 2 M-urea. It was also found that the action of a higher concentration of sodium dodeeyl sulphate (I %, pH 7) on pyocin resulted in complete degradation not only of the core structure but also of the sheath organization, leading to the formation of a 3 s fraction in less than 2 days (Ishii, unpublished work). In the specimen treated with acid (pH 2'4) or alkali (pH Ll-O), also, no intact pyocin particle with the extended sheath was observable. In addition, the distinct behaviours of the two structural components under these conditions should be noted. Extreme fragility of the core under the acidic condition was noticed, in contrast to its considerable resistance to the alkali. While partial disruption of the sheath occurred during the alkali treatment, the whole of its contracted structure seemed to be preserved after the acid treatment, which seems to offer a suitable procedure for the isolation of contracted sheaths. Complete inactivation of pyocin in respect of its bactericidal activity was found by Kageyama & Egami (Hl62) after similar acid. or
FINE STRUCTURE OF A PYOCIN
431
alkali-treatment. They also described the inactivation of pyocin by p-chloromercuribenzoate. Electron micrographs of the pyocin treated with 10 - 3 M-p-chloromercuribenzoate at pH 7·6 were taken in the course of the present study. It was found that 97·2% (989 in 1018) of the pyocin particles possessed contracted sheaths after treatment, whereas the other types of altered particles were not observed. The sheath and the core behave in such different ways under all the chemical treatments mentioned above that their respective subunits are considered to have quite different properties. In addition to these two main components, the structures which may be called base plates and tail fibres can be detected sometimes in electron micrographs of pyocin. The tail fibres are observed on most of the pyocin particles with contracted sheaths in Plate VIII, an electron micrograph of the whole pellet preparation of the mitomycin C-induced cell lysate. However, their presence in the purified specimen is obscure. It is not known yet whether the purification procedure has removed the tail fibres from pyocin, or whether the staining with phosphotungstic acid has damaged them more easily in the purified preparation than in the crude one. Almost all the particles seen in the electron micrograph of the pellet from the lysate (Plate VIII) are interpretable as those ofpyocin. The only exception is a phagelike particle observed in the middle of the Plate. Its round head is accompanied by a bent tail which exhibits no morphological resemblance to pyocin. The phage may correspond to that found by Ikeda, Kageyama & Egami (1964) in the induced cell lysate of the same pyocinogenic strain, R. They have already established that there was no correlation between pyocin and this phage in regard to their immunological behaviour and action spectra. Seaman, Tarmy & Marmur (1964) have studied a group of defective phages, PBSX, produced by mitomycin C-induction in certain strains of Bacillus subtilis. It is worth noticing that PBSX has a characteristic bactericidal activity similar to that of bacteriocins and has no capacity for self-replication in the recipient cell. The electron micrograph revealed that it consisted of a complex mixture of three distinct phage types. Many of the phage particles appeared as ghosts with or without tails, and the detached tails with normal or contracted sheaths were also observed frequently. Our pyocin-forming system, which produces phage tails of a single species almost exclusively, exhibits a striking contrast to the PBSX-forming system. We wish to thank Dr J. Y. Homma, Institute for Infectious Diseases, University of Tokyo, for the generous gift of the bacterial strain and Mr K. Kurita for his skilled assistance. REFERENCES Brenner, S. & Horne, R. W. (1959). Biochim. biophys. Acta, 34, 103. Brenner, S., Streisinger, G., Horne, R. W., Champe, S. P., Barnett, L., Benzer, S. & Rees, M. W. (1959). J. Mol. Bioi. 1, 281. Ikeda, K., Kageyama, M. & Egami, F. (1964). J. Biochem., Tokyo, 55,54. Jacob, F. (1954). Ann. Inst, Pasteur, 86, 149. Kageyama, M. (1964). J. Biochem., Tokyo, 55, 49. Kageyama, M. & Egami, F. (1962). Life Sci. no. 9, 471. Sakata, S. (1958). J. Electronmicroscopy, 6, 75. Seaman, E., Tarrny, E. & Marmur, J. (1964). Biochemistry, 3, 607.
The Fine Structure of a Pyocin SHIN-ICHI ISHII
Department of Biophysics and Biochemistry Faculty of Science, University of Tokyo, Tokyo
y OSHIMI
NISHI
Scientfiic Instruments Department, Naka Works, Hitachi, Ltd., Katsuta, Japan AND FUJIO EGAMI
Department of Biophysics and Biochemistry Faculty of Science, University of Tokyo, Tokyo, Japan (Received 17 May 1965) Negatively stained specimens of pyocin, a bacteriocin produced by strain R Pseudomonas aeruginosa, were examined by electron microscopy, and a close resemblance of the pyocin to T -even bacteriophage tails was demonstrated with regard to their fine structure. The main structure of the pyocin was coneluded to be a double hollow cylinder, 1200 A long and 150 A in outer diameter, which consisted of a "sheath" and a "core". The sheath appeared capable of contraction. The presence of a "base plate" and "tail fibres" was also suggested. The sheath and the core were found to show markedly different behaviour under various chemical treatments, including those by urea, sodium dodecyl sulphate, acid and alkali. They may be composed of protein subunits which differ from each other in chemical properties.
1. Introduction Jacob (1954) was the first to describe pyocin, an antibacterial substance which was produced by certain strains of Pseudomonas aeruginosa and exerted a specific bactericidal activity typical of bacteriocins. Kageyama & Egami (1962) and Kageyama (1964) have recently succeeded in purifying a pyocin as a homogeneous protein of high molecular weight, from the lysate of ultraviolet- or mitomycin C-induced cells of Ps. aeruqinosa strain R. The purified preparation of pyocin, which was shadowed with chromium, appeared in the electron microscope as rod-like particles with a uniform size. They thought the rods resembled bacteriophage tails. In the present study, the use of the negative staining technique (Brenner & Horne, 1959) led to the elucidation of the fine structure of the pyocin. The presence of at least two structural components, "sheath" and "core", was demonstrated, as in the tails of T-even phages. The behaviour of these structural components under various chemical treatments has also been examined.
2. Materials and Methods The purified pyocin was prepared from the lysate of mitomycin C-induced cells of Ps, aeruginosa strain R by the procedure of Kageyama (1964). Either directly or after various chemical treatments, the pyocin in 0·1 M-NaCl containing 0·005 M-tris buffer (pH 7-2),
428
PLATE
1. An electron micrograph of purified pyocin ( X 250,000).
[facing p. 430
PLATE II. (a) A micrograph at a higher magnification of the same specimen shown in Plate I (X 400,000). (b) Pyocin treated with mild alkali (pH 10,5). Three sets of striations (in directions indicated with arrows) are visible on the surface of an extended sheath (x 400,000). (c) A micrograph at a higher magnification of the same specimen shown in Plate VIII. The contracted sheath appears to accompany a base plate and tail fibres (x 400,000). (d) A micrograph at a higher magnification of the same specimen shown in Plate IV ( X 400,000).
PLATE III. A micrograph of pyocin treated with 0·1 % sodium dodecyl sulphate showing the formation of stretched helical structure from ends of partially disintegrated sheaths. Cores seem to be decomposed completely ( X 250,000).
loooA PLATE IV. Pyocin treated with 6 llil-urea. A number of sheaths or their segments can be seen standing upright, revealing the "cog wheel" arrangement of the subunits ( X 250.000).
PLATE V. Pyocin treated with acid (pH 2,4). The whole of the contracted sheath structure seems to be preserved, while complete disappearance of the core structure occurs also in this case ( X 250,000).
PLATE VI. Pyocin treated under slightly acidic conditions (pH 6). Many small rings may correspond to the sections of disrupted cores seen end-on ( X 250,000).
PLATE VII. Structures resulting from the treatment of pyocin with alkali (pH U·O). These consist of contracted sheaths with or without cores, and detached cores. The sheaths demonstrate disrupted figures frequently (x 250,000).
PLATE VIII. A whole pellet preparation from the induced-cell lysate. Almost all the particles are interpretable as those of pyocin, except a phage-like particle observed in the middle of the field ( X 250,000).
FINE STRUCTURE OF A PYOCIN
429
was diluted five- to tenfold, usually with distilled water, to afford the sample solution of appropriate concentration (0,1 to 0·2 mg proteinjml.) for negative staining. A crude pellet preparation of the lysate was obtained by the following process. The mitomycin O-induced cell lysate (35 ml.) was first treated with I /kg/ml. deoxyribonuclease (Worthington) for 60 min at 37°0 and was cleared of bacterial debris by low-speed centrifugation. The supernatant fraction was centrifuged in the type 40P Hitachi ultracentrifuge for 45 min at 25,000 rev Jrou: (about 60,000 g). The pellet was suspended in 5 ml. of 0·05 M-tris buffer (pH 7,7). and used for the electron microscope investigation. Negatively stained specimens were prepared by depositing a drop of the sample solutions on a micro-grid (Sakata, 1958) and adding another drop of a 2% solution of phosphotungstic acid adjusted to pH 7·0 with potassium hydroxide. The excess solution was withdrawn by touching the edge of the grid with filter paper. The grids were always mounted in the electron microscope with the specimen side away from the electron source, and photographic enlargements were made from the electron microscope plates by inserting them in the enlarger with the emulsion side away from the light source. The type HU-IIA Hitachi electron microscope was used at a magnification of 50,000.
3. Results Plate I is an electron micrograph of the purified pyocin. There are rod-like particles, 1200 A long and 150 A wide, which show striations running nearly at right angles to the particle axes. Their appearance is very similar to that of the T-even bacteriophage tails (Brenner et al., 1959). In addition, particles with "contracted sheaths" are observed in the Plate. "Cores" are missing in some of the particles of this type. The profiles of these contracted sheaths can be interpreted as hollow cylinders 460 A long and 180 A in outer diameter. The core also shows a hollow tubular structure 1200 A long and 57 A in outer diameter. The micrograph Plate II(a) indicates two typical pyocin particles with the extended sheaths at a higher enlargement, where approximately 35 regular striations can be counted along their lengths. Plate II(b) is a selected area of an electron micrograph of the pyocin treated under mild alkaline conditions (pH 10,5) for five hours. In the middle of this Plate there is a representative of the pyocin particles which still have extended sheaths after this treatment. There are two more sets of striations clearly visible at opposite slanting angles (indicated by arrows A and B) on the surface of this particle, besides the cross-striations (arrow C) mentioned above. The fine round particles detectable along these three families of striations may eventually be correlated with the protein subunits which constitute the whole sheath. The "base plate" structure is also visible at a distal end of the pyocin particle in Plate II(b). The contracted sheath in Plate II(c) appears to have pulled up the base plate leaving an end of the core free, and the base plate seems to accompany "tail fibres" . Plate III demonstrates characteristic structures produced by treating pyocin with 0·1 % sodium dodecyl sulphate at pH 7 for about 20 hours. Contracted sheaths have been partly disintegrated, releasing stretched helices from their ends, while all cores have disappeared. The appearance of the stretched helices suggests that a dominant helical array of the sheath subunits is present along a set of the cross-striations observed on the extended sheath. Treatment with 6 M-urea also decomposes the core structures completely and leaves empty contracted sheaths as seen in Plate IV. Partial disruption of the sheaths occurs, but no stretching of the helical array is observed in this case. There are several sheaths or their disrupted segments standing upright. They display the "cog wheel" appearance similar to that described by
430
S. ISHII, Y. NISHI AND F. EGAMI
Brenner et at. (1959) about the contracted sheaths of T2 phage tails. Their enlarged images are shown in Plate II(d). In the case of acid treatment (0-05 N-acetic + 0·05 N-formic acid (pH 2-4), 20 hours), the entire contracted sheath structure appears to be preserved as shown in Plate V, while cores are destroyed completely again. Plate VI, a micrograph of the \ pyocin treated under a slightly acidic condition (pH 6, about 20 hours), exhibits many small rings which may correspond to the sections of disrupted cores seen end-on. A double-ring (indicated with an arrow in Plate VI) is thought to be an end-on view of the contracted sheath still accommodating a piece of the disrupted core. Plate VII shows that alkali-treatment (0-05 M-Na2C03 (pH 11-0), 20 hours) of pyocin produced a variety of altered particles. All the sheaths have been contracted and some detached from cores. In contrast with the cores, the sheaths demonstrate disrupted figures quite frequently. Many sheaths, probably disrupted, are observed end-on, showing a cog wheel appearance again.
4. Discussion Evidence is presented to demonstrate morphological resemblance of pyocin produced by Ps. aeruginosa strain R to T-even phage tails. The main structure of the pyocin is concluded to be a double hollow cylinder which consists of a sheath and a core. The sheath, which seems to be capable of contraction, appears to be composed of numerous protein subunits arranged in screw symmetry (see Plate II(a), (b). It can further be argued that a fundamental helical array of the sheath subunits along the cross-striations (arrow C in Plate II(b» has produced the stretched spirals seen in the specimen treated with 0-1 % sodium dodecyl sulphate (Plate III). The number of the cross-striations is about 35 and these may represent the number of turns of the helix in the extended sheath. On the other hand, about 12 "cogs" can be seen in end-on view of contracted sheaths (Plate II(d». This is not likely to be the number of subunits in one turn of the helix in the extended structure, however, because the increase in diameter of the sheath at the time of contraction suggests that this process accompanies an increase in the number of subunits per turn, as already discussed by Brenner et at. (1959). Chemical studies on purified contracted sheaths, now in progress, are expected to lead to more precise information on all the subunits in the sheath. The structure of cores in pyocin particles is found to be very labile upon treatment with urea (6 M) or sodium dodecyl sulphate (0-1 %). Complete disappearance of the core structure in electron micrographs was seen even with the specimen treated with 2 M-urea. It was also found that the action of a higher concentration of sodium dodeeyl sulphate (I %, pH 7) on pyocin resulted in complete degradation not only of the core structure but also of the sheath organization, leading to the formation of a 3 s fraction in less than 2 days (Ishii, unpublished work). In the specimen treated with acid (pH 2'4) or alkali (pH Ll-O), also, no intact pyocin particle with the extended sheath was observable. In addition, the distinct behaviours of the two structural components under these conditions should be noted. Extreme fragility of the core under the acidic condition was noticed, in contrast to its considerable resistance to the alkali. While partial disruption of the sheath occurred during the alkali treatment, the whole of its contracted structure seemed to be preserved after the acid treatment, which seems to offer a suitable procedure for the isolation of contracted sheaths. Complete inactivation of pyocin in respect of its bactericidal activity was found by Kageyama & Egami (Hl62) after similar acid. or
FINE STRUCTURE OF A PYOCIN
431
alkali-treatment. They also described the inactivation of pyocin by p-chloromercuribenzoate. Electron micrographs of the pyocin treated with 10 - 3 M-p-chloromercuribenzoate at pH 7·6 were taken in the course of the present study. It was found that 97·2% (989 in 1018) of the pyocin particles possessed contracted sheaths after treatment, whereas the other types of altered particles were not observed. The sheath and the core behave in such different ways under all the chemical treatments mentioned above that their respective subunits are considered to have quite different properties. In addition to these two main components, the structures which may be called base plates and tail fibres can be detected sometimes in electron micrographs of pyocin. The tail fibres are observed on most of the pyocin particles with contracted sheaths in Plate VIII, an electron micrograph of the whole pellet preparation of the mitomycin C-induced cell lysate. However, their presence in the purified specimen is obscure. It is not known yet whether the purification procedure has removed the tail fibres from pyocin, or whether the staining with phosphotungstic acid has damaged them more easily in the purified preparation than in the crude one. Almost all the particles seen in the electron micrograph of the pellet from the lysate (Plate VIII) are interpretable as those ofpyocin. The only exception is a phagelike particle observed in the middle of the Plate. Its round head is accompanied by a bent tail which exhibits no morphological resemblance to pyocin. The phage may correspond to that found by Ikeda, Kageyama & Egami (1964) in the induced cell lysate of the same pyocinogenic strain, R. They have already established that there was no correlation between pyocin and this phage in regard to their immunological behaviour and action spectra. Seaman, Tarmy & Marmur (1964) have studied a group of defective phages, PBSX, produced by mitomycin C-induction in certain strains of Bacillus subtilis. It is worth noticing that PBSX has a characteristic bactericidal activity similar to that of bacteriocins and has no capacity for self-replication in the recipient cell. The electron micrograph revealed that it consisted of a complex mixture of three distinct phage types. Many of the phage particles appeared as ghosts with or without tails, and the detached tails with normal or contracted sheaths were also observed frequently. Our pyocin-forming system, which produces phage tails of a single species almost exclusively, exhibits a striking contrast to the PBSX-forming system. We wish to thank Dr J. Y. Homma, Institute for Infectious Diseases, University of Tokyo, for the generous gift of the bacterial strain and Mr K. Kurita for his skilled assistance. REFERENCES Brenner, S. & Horne, R. W. (1959). Biochim. biophys. Acta, 34, 103. Brenner, S., Streisinger, G., Horne, R. W., Champe, S. P., Barnett, L., Benzer, S. & Rees, M. W. (1959). J. Mol. Bioi. 1, 281. Ikeda, K., Kageyama, M. & Egami, F. (1964). J. Biochem., Tokyo, 55,54. Jacob, F. (1954). Ann. Inst, Pasteur, 86, 149. Kageyama, M. (1964). J. Biochem., Tokyo, 55, 49. Kageyama, M. & Egami, F. (1962). Life Sci. no. 9, 471. Sakata, S. (1958). J. Electronmicroscopy, 6, 75. Seaman, E., Tarrny, E. & Marmur, J. (1964). Biochemistry, 3, 607.