Proc. Natl. Acad. Sci. USA
Vol. 75, No. 7, pp. 3206-3210, July 1978 Biochemistry
Visualization of the intracellular development of bacteriophage X, with special reference to DNA packaging (electron microscopy/osmotically ruptured cell/DNA-precursor head complex/head mutants/continuous packaging)
HIDEO YAMAGISHI AND MITSUMASA OKAMOTO Department of Biophysics, Faculty of Science, Kyoto University, Kyoto 606, Japan
Communicated by Dale Kaiser, May 1, 1978
ABSTRACT To reveal intermediates in X DNA packaging, infected cells were osmotically ruptured and the cell lysates were deposited on electron microscope grids by sedimentation through a sucrose/formalin cushion. A fixation procedure that crosslinks head-related structures to DNA allowed us to study successive stages in the process of head filling. Three types of head-related structures can be distinguished: (i) empty heads (petit X), less angular in outline than complete X heads; (ii) heads partially filled with DNA (partially filled heads), having a roundish outline; and (iii) particles tightly packed with DNA (full heads), having a hexagonal outline. DNA-head complexes were bound either at the terminal end of a DNA thread or at a point intermediate along the thread. The terminal complexes were more abundant. No head-related structures could be found in an induced X mutant lysogen blocked in the synthesis of petit X (amber in X gene E). One type of mutant blocked in DNA packaging (amber in gene A) produces empty heads and free tails, whereas another (amber in gene D) produces partially filled heads in addition. Our data suggest that a DNA-petit A complex may be an early intermediate in packaging and that the A DNA substrate can be a cohesive-ended concatemer or a concatemer with double-stranded cohesive site sequences.
heat-inducible lysogen, 594(XEam4cI857Sam7), was prepared in this work. Media and Chemicals. X broth (9) was filtered through a 0.45-Mm Millipore filter to eliminate insoluble materials that might deposit on the electron microscope grid. A solution of 10% formalin and 0.1 M sucrose was prepared with neutral formalin (Merck) and adjusted to pH 8.5 with borate buffer (pH 10). The formalin/sucrose solution, dilute Kodak Photo-Flo solution, and phosphotungstic acid (PTA) solution were filtered through a 0.2-Mm membrane filter (Sartorius) before each use (7). T4 lysozyme was a gift from E. Akaboshi. Conditions of Phage Growth. To enrich intracellular phage DNA after infection, host DNA synthesis was inhibited by UV light irradiation [RecA - bacteria are extremely sensitive to low UV doses (10, 11)]. 594recA41 was grown, by shaking at 370, to a concentration of about 4 X 108 cells per ml in X broth. Five milliliters of the culture was placed in a petri dish (15 cm diameter) and irradiated with a low dose (440 erg/mm2) or with a high dose (2200 erg/mm2) (1 erg = 1 X 10-7 joule). The cells were collected by centrifugation and immediately suspended in 0.1 vol of 0.02 M MgSO4. The cell suspension was infected with XcI857Sam7 at a multiplicity of 5. After 10-min incubation at 370, infected cells were diluted 1:10 (normal cell density) or 1:3 (high cell density) with X broth and shaken at 37°. The plaque-forming ability of infected cells was not significantly reduced by the UV irradiation but a 3-fold increase in cell density caused a 3-fold decrease in average burst size. To facilitate the recognition of DNA-head complexes in the electron microscope, a burst size of less than 100 is preferable. Therefore, we lysed cells infected with wild-type X at an early stage (60 min after infection) or at high cell density. The cells infected with mutant X were lysed 120 min after infection. To confirm the absence of petit X particles, an E- mutant lysogen was grown at 320 to 4 X 108 cells per ml. Phage production was initiated by incubating the cultures at 430 for 15 min with continuous aeration, after which the cultures were aerated at 370 for 120 min. Lysis Procedures and Electron Microscopy. The basic procedures, described in detail elsewhere (7), have been somewhat modified. Our current procedure is as follows. (i) A 10-All sample of the culture is made hypertonic by 1:10 dilution into 0.1 M Tris/0. 1 M NaCI/20% (wt/vol) sucrose, pH 7.8, at 00. A 0.5-Ml sample of T4 lysozyme solution (2 mg/ml) is added to 100 Al of the hypertonic cell suspension and the mixture is kept on crushed ice for 3 min. By this treatment, bacterial cells are rendered osmotically sensitive. (ii) The Lucite microcentrifugation chamber, 4 mm in diameter and 6 mm in depth, is filled with a solution of 10%
A head morphogenesis can be divided into two stages: (i) building an empty head and (ii) encapsidation of the phage DNA into that empty head (for a review, see ref. 1). Although electron microscopic studies of thin sections of X-infected bacteria have revealed some of the intracellular process of phage assembly (2-4), DNA is not visible in thin sections as an extended single thread. Consequently, the details of packaging and maturation of A DNA have been obscure. This communication describes experiments performed to gain direct information about the pathways through which A DNA is condensed into a preformed empty head. Our efforts to visualize the process of head filling have profited by the techniques of rapid isolation of genetic material from cells, as developed by Miller and his coworkers (5-7) and the techniques of rotary shadowing with platinum.
MATERIALS AND METHODS Bacterial Strain and Bacteriophage. Escherichia coli 594recA41 was obtained from H. Ogawa. Bacteriophage carrying the mutation for lysis inhibition, XcI857Sam7 (8), was used as "wild type." Defective lysogens W3101(XAam32cI857Sam7) and W3101(ADaml5c1857Sam7) were gifts from I. Katsura. Stocks of both mutant phages were prepared by superinfection with helper phage and temperature induction. XEam4cI857Sam7 was constructed by crossing AEam4 and Xc1857Sam7. AEam4 was originally received from A. Campbell. Their genotypes were confirmed by complementation tests. A The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "
Vol. 75, No. 7, pp. 3206-3210, July 1978 Biochemistry
Visualization of the intracellular development of bacteriophage X, with special reference to DNA packaging (electron microscopy/osmotically ruptured cell/DNA-precursor head complex/head mutants/continuous packaging)
HIDEO YAMAGISHI AND MITSUMASA OKAMOTO Department of Biophysics, Faculty of Science, Kyoto University, Kyoto 606, Japan
Communicated by Dale Kaiser, May 1, 1978
ABSTRACT To reveal intermediates in X DNA packaging, infected cells were osmotically ruptured and the cell lysates were deposited on electron microscope grids by sedimentation through a sucrose/formalin cushion. A fixation procedure that crosslinks head-related structures to DNA allowed us to study successive stages in the process of head filling. Three types of head-related structures can be distinguished: (i) empty heads (petit X), less angular in outline than complete X heads; (ii) heads partially filled with DNA (partially filled heads), having a roundish outline; and (iii) particles tightly packed with DNA (full heads), having a hexagonal outline. DNA-head complexes were bound either at the terminal end of a DNA thread or at a point intermediate along the thread. The terminal complexes were more abundant. No head-related structures could be found in an induced X mutant lysogen blocked in the synthesis of petit X (amber in X gene E). One type of mutant blocked in DNA packaging (amber in gene A) produces empty heads and free tails, whereas another (amber in gene D) produces partially filled heads in addition. Our data suggest that a DNA-petit A complex may be an early intermediate in packaging and that the A DNA substrate can be a cohesive-ended concatemer or a concatemer with double-stranded cohesive site sequences.
heat-inducible lysogen, 594(XEam4cI857Sam7), was prepared in this work. Media and Chemicals. X broth (9) was filtered through a 0.45-Mm Millipore filter to eliminate insoluble materials that might deposit on the electron microscope grid. A solution of 10% formalin and 0.1 M sucrose was prepared with neutral formalin (Merck) and adjusted to pH 8.5 with borate buffer (pH 10). The formalin/sucrose solution, dilute Kodak Photo-Flo solution, and phosphotungstic acid (PTA) solution were filtered through a 0.2-Mm membrane filter (Sartorius) before each use (7). T4 lysozyme was a gift from E. Akaboshi. Conditions of Phage Growth. To enrich intracellular phage DNA after infection, host DNA synthesis was inhibited by UV light irradiation [RecA - bacteria are extremely sensitive to low UV doses (10, 11)]. 594recA41 was grown, by shaking at 370, to a concentration of about 4 X 108 cells per ml in X broth. Five milliliters of the culture was placed in a petri dish (15 cm diameter) and irradiated with a low dose (440 erg/mm2) or with a high dose (2200 erg/mm2) (1 erg = 1 X 10-7 joule). The cells were collected by centrifugation and immediately suspended in 0.1 vol of 0.02 M MgSO4. The cell suspension was infected with XcI857Sam7 at a multiplicity of 5. After 10-min incubation at 370, infected cells were diluted 1:10 (normal cell density) or 1:3 (high cell density) with X broth and shaken at 37°. The plaque-forming ability of infected cells was not significantly reduced by the UV irradiation but a 3-fold increase in cell density caused a 3-fold decrease in average burst size. To facilitate the recognition of DNA-head complexes in the electron microscope, a burst size of less than 100 is preferable. Therefore, we lysed cells infected with wild-type X at an early stage (60 min after infection) or at high cell density. The cells infected with mutant X were lysed 120 min after infection. To confirm the absence of petit X particles, an E- mutant lysogen was grown at 320 to 4 X 108 cells per ml. Phage production was initiated by incubating the cultures at 430 for 15 min with continuous aeration, after which the cultures were aerated at 370 for 120 min. Lysis Procedures and Electron Microscopy. The basic procedures, described in detail elsewhere (7), have been somewhat modified. Our current procedure is as follows. (i) A 10-All sample of the culture is made hypertonic by 1:10 dilution into 0.1 M Tris/0. 1 M NaCI/20% (wt/vol) sucrose, pH 7.8, at 00. A 0.5-Ml sample of T4 lysozyme solution (2 mg/ml) is added to 100 Al of the hypertonic cell suspension and the mixture is kept on crushed ice for 3 min. By this treatment, bacterial cells are rendered osmotically sensitive. (ii) The Lucite microcentrifugation chamber, 4 mm in diameter and 6 mm in depth, is filled with a solution of 10%
A head morphogenesis can be divided into two stages: (i) building an empty head and (ii) encapsidation of the phage DNA into that empty head (for a review, see ref. 1). Although electron microscopic studies of thin sections of X-infected bacteria have revealed some of the intracellular process of phage assembly (2-4), DNA is not visible in thin sections as an extended single thread. Consequently, the details of packaging and maturation of A DNA have been obscure. This communication describes experiments performed to gain direct information about the pathways through which A DNA is condensed into a preformed empty head. Our efforts to visualize the process of head filling have profited by the techniques of rapid isolation of genetic material from cells, as developed by Miller and his coworkers (5-7) and the techniques of rotary shadowing with platinum.
MATERIALS AND METHODS Bacterial Strain and Bacteriophage. Escherichia coli 594recA41 was obtained from H. Ogawa. Bacteriophage carrying the mutation for lysis inhibition, XcI857Sam7 (8), was used as "wild type." Defective lysogens W3101(XAam32cI857Sam7) and W3101(ADaml5c1857Sam7) were gifts from I. Katsura. Stocks of both mutant phages were prepared by superinfection with helper phage and temperature induction. XEam4cI857Sam7 was constructed by crossing AEam4 and Xc1857Sam7. AEam4 was originally received from A. Campbell. Their genotypes were confirmed by complementation tests. A The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "
