Identification of a Morphogenic Intermediate of the Bacteriophage Mu Baseplate
Affiliation(s)
Faculty of Science and Technology, Division of Molecular Science, Gunma University, Gunma, Japan.
Faculty of Science and Technology, Division of Molecular Science, Gunma University, Gunma, Japan.
ABSTRACT
Bacteriophage morphogenesis is a model system for investigating sequential molecular assembly. The Mu phage is one of the most classical Myoviridae. Although it is well known as a mobile genetic element, the details of its morphogenesis remain unclear. Analysis of conditional lethal mutants and genome analysis of the Mu phage have suggested that genes 42, 43, 44, 45, 46, 47, and 48 are essential for its baseplate assembly. Since we have already reported X-ray structures of the products of genes 44 (gp44) and 45 (gp45), we here tried to purify the remaining Mu phage baseplate subunits, gp42, gp43, gp46, gp47, and gp48, to investigate the baseplate assembly process. In the case of gp42 expression, the transformed E. coli cells showed growth inhibition after induction and no gp42 fractions were observed. However, gp43, gp46, gp47, and gp48 were successfully expressed and purified, although gp48 could not be applied to further analysis, because the amount of soluble fraction was very low. Based on analytical ultracentrifugation, we concluded that gp43 formed a monomer, gp46 was a monomer, and gp47 occurred as both a monomer and dimer in solution. Moreover, we found that gp43 and gp45 formed an intermediate complex in the baseplate assembly process.
Bacteriophage morphogenesis is a model system for investigating sequential molecular assembly. The Mu phage is one of the most classical Myoviridae. Although it is well known as a mobile genetic element, the details of its morphogenesis remain unclear. Analysis of conditional lethal mutants and genome analysis of the Mu phage have suggested that genes 42, 43, 44, 45, 46, 47, and 48 are essential for its baseplate assembly. Since we have already reported X-ray structures of the products of genes 44 (gp44) and 45 (gp45), we here tried to purify the remaining Mu phage baseplate subunits, gp42, gp43, gp46, gp47, and gp48, to investigate the baseplate assembly process. In the case of gp42 expression, the transformed E. coli cells showed growth inhibition after induction and no gp42 fractions were observed. However, gp43, gp46, gp47, and gp48 were successfully expressed and purified, although gp48 could not be applied to further analysis, because the amount of soluble fraction was very low. Based on analytical ultracentrifugation, we concluded that gp43 formed a monomer, gp46 was a monomer, and gp47 occurred as both a monomer and dimer in solution. Moreover, we found that gp43 and gp45 formed an intermediate complex in the baseplate assembly process.
KEYWORDS
Bacteriophage, Baseplate, Self-Assembly, Assembly Intermediate, Analytical Ultracentrifugation
Bacteriophage, Baseplate, Self-Assembly, Assembly Intermediate, Analytical Ultracentrifugation
Cite this paper
Tsukamoto, N. , Kanazawa, Y. , Shimamori, Y. , Yoshida, K. and Takeda, S. (2014) Identification of a Morphogenic Intermediate of the Bacteriophage Mu Baseplate. Advances in Microbiology, 4, 1155-1163. doi: 10.4236/aim.2014.416125.
Tsukamoto, N. , Kanazawa, Y. , Shimamori, Y. , Yoshida, K. and Takeda, S. (2014) Identification of a Morphogenic Intermediate of the Bacteriophage Mu Baseplate. Advances in Microbiology, 4, 1155-1163. doi: 10.4236/aim.2014.416125.
References
[1] Epstein, R.H., Bolle, A., Steinberg, C.M., Kellenberger, E., Boy de la Tour, E., Chevalley, R. , Edgar, R.S., Susman, M., Denhard, G.H. and Lialausis, A. (1963) Physiological Studies of Conditional Lethal Mutants of Bacteriophage T4D. Cold Spring Harbor Symposia on Quantitative Biology, 28, 375-394.
http://dx.doi.org/10.1101/SQB.1963.028.01.053 [2] Kikuchi, Y. and King, J. (1975) Genetic Control of Bacteriophage T4 Baseplate Morphogenesis. I. Sequential Assembly of the Major Precursor, in Vivo and in Vitro. Journal of Molecular Biology, 99, 645-672. http://dx.doi.org/10.1016/S0022-2836(75)80178-1 [3] Kikuchi, Y. and King, J. (1975) Genetic Control of Bacteriophage T4 Baseplate Morphogenesis. II. Mutants Unable to Form the Central Part of the Baseplate. Journal of Molecular Biology, 99, 673-694.
http://dx.doi.org/10.1016/S0022-2836(75)80179-3 [4] Kikuchi, Y. and King, J. (1975) Genetic Control of Bacteriophage T4 Baseplate Morphogenesis. III. Formation of the Central Plug and Overall Assembly Pathway. Journal of Molecular Biology, 99, 695-716. http://dx.doi.org/10.1016/S0022-2836(75)80180-X [5] Wood, W.B., Edgar, R.S., King, J., Lielausis, I. and Henninger, M. (1968) Bacteriophage Assembly. Federation Proceedings, 27, 1160-1166. [6] Aksyuk, A.A. and Rossmann, M.G. (2011) Bacteriophage Assembly. Viruses, 3, 172-203.
http://dx.doi.org/10.3390/v3030172 [7] Yap, M.L., Mio, K., Leiman, P.G., Kanamaru, S. and Arisaka, F. (2010) The Baseplate Wedges of Bacteriophage T4 Spontaneously Assemble into Hubless Baseplate-Like Structure in Vitro. Journal of Molecular Biology, 395, 349-360. http://dx.doi.org/10.1016/j.jmb.2009.10.071 [8] Leiman, P.G., Arisaka, F., Van Raaij, M.J., Kostyuchenko, V.A., Aksyuk, A.A., Kanamaru, S. and Rossmann, M.G. (2010) Morphogenesis of the T4 Tail and Tail Fibers. Virology Journal, 7, 355.
http://dx.doi.org/10.1186/1743-422X-7-355 [9] Kanamaru, S., Leiman, P.G., Kostyuchenko, V.A., Chipman, P.R., Mesyanzhinov, V.V., Arisaka, F. and Rossmann, M.G. (2002) Structure of the Cell-Puncturing Device of Bacteriophage T4. Nature, 415, 553-557. http://dx.doi.org/10.1038/415553a [10] Bebeacua, C., Lai, L., Vegge, C.S., Brondsted, L., Van Heel, M., Veesler, D. and Cambillau, C. (2013) Visualizing a Complete Siphoviridae Member by Single-Particle Electron Microscopy: The Structure of Lactococcal Phage TP901-1. Journal of Virology, 87, 1061-1068.
http://dx.doi.org/10.1128/JVI.02836-12 [11] Badasso, M.O., Leiman, P.G., Tao, Y., He, Y., Ohlendorf, D.H., Rossmann, M.G. and Anderson, D. (2000) Purification, Crystallization and Initial X-Ray Analysis of the Head-Tail Connector of Bacteriophage phi29. Acta crystallographica Section D, Biological Crystallography, 56, 1187-1190. http://dx.doi.org/10.1107/S0907444900009239 [12] Tang, L., Marion, W.R., Cingolani, G., Prevelige, P.E. and Johnson, J.E. (2005) Three-Dimensional Structure of the Bacteriophage P22 Tail Machine. The EMBO Journal, 24, 2087-2095. http://dx.doi.org/10.1038/sj.emboj.7600695 [13] Fokine, A., Zhang, Z.H., Kanamaru, S., Bowman, V.D., Aksyuk, A.A., Arisaka, F., Rao, V.B. and Rossmann, M.G. (2013) The Molecular Architecture of the Bacteriophage T4 Neck. Journal of Molecular Biology, 425, 1731-1744. http://dx.doi.org/10.1016/j.jmb.2013.02.012 [14] Bebeacua, C., Tremblay, D., Farenc, C., Chapot-Chartier, M.P., Sadovskaya, I., Van Heel, M., Veesler, D., Moineau, S. and Cambillau, C. (2013) Structure, Adsorption to Host, and Infection Mechanism of Virulent Lactococcal Phage p2. Journal of Virology, 87, 12302-12312.
http://dx.doi.org/10.1128/JVI.02033-13 [15] Xu, J., Hendrix, R.W. and Duda, R.L. (2013) A Balanced Ratio of Proteins from Gene G and Frameshift-Extended Gene GT Is Required for Phage Lambda Tail Assembly. Journal of Molecular Biology, 425, 3476-3487. http://dx.doi.org/10.1016/j.jmb.2013.07.002 [16] Hendrix, R.W. and Johnson, J.E. (2012) Bacteriophage HK97 Capsid Assembly and Maturation. Advances in Experimental Medicine and Biology, 726, 351-363.
http://dx.doi.org/10.1007/978-1-4614-0980-9_15 [17] Arisaka, F. (2005) Assembly and Infection Process of Bacteriophage T4. Chaos, 15, Article ID: 047502. http://dx.doi.org/10.1063/1.2142136 [18] O’Day, K., Schultz, D., Ericsen, W., Rawluk, L. and Howe, M. (1979) Correction and Refinement of the Genetic Map of Bacteriophage Mu. Virology, 93, 320-328.
http://dx.doi.org/10.1016/0042-6822(79)90236-8 [19] Giphart-Gassler, M., Wijffelman, C. and Reeve, J. (1981) Structural Polypeptides and Products of Late Genes of Bacteriophage Mu: Characterization and Functional Aspects. Journal of Molecular Biology, 145, 139-163. http://dx.doi.org/10.1016/0022-2836(81)90338-7 [20] Grundy, F.J. and Howe, M.M. (1985) Morphogenetic Structures Present in Lysates of Amber Mutants of Bacteriophage Mu. Virology, 143, 485-504. http://dx.doi.org/10.1016/0042-6822(85)90388-5 [21] Morgan, G.J., Hatfull, G.F., Casjens, S. and Hendrix, R.W. (2002) Bacteriophage Mu Genome Sequence: Analysis and Comparison with Mu-Like Prophages in Haemophilus, Neisseria and Deinococcus. Journal of Molecular Biology, 317, 337-359. http://dx.doi.org/10.1006/jmbi.2002.5437 [22] Kondou, Y., Kitazawa, D., Takeda, S., Tsuchiya, Y., Yamashita, E., Mizuguchi, M., Kawano, K. and Tsukihara, T. (2005) Structure of the Central Hub of Bacteriophage Mu Baseplate Determined by X-Ray Crystallography of gp44. Journal of Molecular Biology, 352, 976-985.
http://dx.doi.org/10.1016/j.jmb.2005.07.044 [23] Harada, K., Yamashita, E., Nakagawa, A., Miyafusa, T., Tsumoto, K., Ueno, T., Toyama, Y. and Takeda, S. (2013) Crystal Structure of the C-Terminal Domain of Mu Phage Central Spike and Functions of Bound Calcium Ion. Biochimica et Biophysica Acta, 1834, 284-291.
http://dx.doi.org/10.1016/j.bbapap.2012.08.015 [24] Kitazawa, D., Takeda, S., Kageyama, Y., Tomihara, M. and Fukada, H. (2005) Expression and Characterization of a Baseplate Protein for Bacteriophage Mu, gp44. Journal of Biochemistry, 137, 601-606. http://dx.doi.org/10.1093/jb/mvi076 [25] Schuck, P. (2000) Size-Distribution Analysis of Macromolecules by Sedimentation Velocity Ultracentrifugation and Lamm Equation Modeling. Biophysical Journal, 78, 1606-1619.
http://dx.doi.org/10.1016/S0006-3495(00)76713-0 [26] Schuck, P., Perugini, M.A., Gonzales, N.R., Howlett, G.J. and Schubert, D. (2002) Size-Distribution Analysis of Proteins by Analytical Ultracentrifugation: Strategies and Application to Model Systems. Biophysical Journal, 82, 1096- 1111. http://dx.doi.org/10.1016/S0006-3495(02)75469-6 [27] Lebowitz, J., Lewis, M.S. and Schuck, P. (2002) Modern Analytical Ultracentrifugation in Protein Science: A Tutorial Review. Protein Science, 11, 2067-2079. http://dx.doi.org/10.1110/ps.0207702 [28] Suzuki, H., Yamada, S., Toyama, Y. and Takeda, S. (2010) The C-Terminal Domain Is Sufficient for Host-Binding Activity of the Mu Phage Tail-Spike Protein. Biochimica et Biophysica Acta, 1804, 1738-1742. http://dx.doi.org/10.1016/j.bbapap.2010.05.003 [29] Piuri, M. and Hatfull, G.F. (2006) A Peptidoglycan Hydrolase Motif within the Mycobacteriophage TM4 Tape Measure Protein Promotes Efficient Infection of Stationary Phase Cells. Molecular Microbiology, 62, 1569-1585. http://dx.doi.org/10.1111/j.1365-2958.2006.05473.x [30] Rodriguez-Rubio, L., Gutierrez, D., Martinez, B., Rodriguez, A., Gotz, F. and Garcia, P. (2012) The Tape Measure Protein of the Staphylococcus aureus Bacteriophage vB_SauS-phiIPLA35 Has an Active Muramidase Domain. Applied and Environmental Microbiology, 78, 6369-6371.
http://dx.doi.org/10.1128/AEM.01236-12
[1] Epstein, R.H., Bolle, A., Steinberg, C.M., Kellenberger, E., Boy de la Tour, E., Chevalley, R. , Edgar, R.S., Susman, M., Denhard, G.H. and Lialausis, A. (1963) Physiological Studies of Conditional Lethal Mutants of Bacteriophage T4D. Cold Spring Harbor Symposia on Quantitative Biology, 28, 375-394.
http://dx.doi.org/10.1101/SQB.1963.028.01.053 [2] Kikuchi, Y. and King, J. (1975) Genetic Control of Bacteriophage T4 Baseplate Morphogenesis. I. Sequential Assembly of the Major Precursor, in Vivo and in Vitro. Journal of Molecular Biology, 99, 645-672. http://dx.doi.org/10.1016/S0022-2836(75)80178-1 [3] Kikuchi, Y. and King, J. (1975) Genetic Control of Bacteriophage T4 Baseplate Morphogenesis. II. Mutants Unable to Form the Central Part of the Baseplate. Journal of Molecular Biology, 99, 673-694.
http://dx.doi.org/10.1016/S0022-2836(75)80179-3 [4] Kikuchi, Y. and King, J. (1975) Genetic Control of Bacteriophage T4 Baseplate Morphogenesis. III. Formation of the Central Plug and Overall Assembly Pathway. Journal of Molecular Biology, 99, 695-716. http://dx.doi.org/10.1016/S0022-2836(75)80180-X [5] Wood, W.B., Edgar, R.S., King, J., Lielausis, I. and Henninger, M. (1968) Bacteriophage Assembly. Federation Proceedings, 27, 1160-1166. [6] Aksyuk, A.A. and Rossmann, M.G. (2011) Bacteriophage Assembly. Viruses, 3, 172-203.
http://dx.doi.org/10.3390/v3030172 [7] Yap, M.L., Mio, K., Leiman, P.G., Kanamaru, S. and Arisaka, F. (2010) The Baseplate Wedges of Bacteriophage T4 Spontaneously Assemble into Hubless Baseplate-Like Structure in Vitro. Journal of Molecular Biology, 395, 349-360. http://dx.doi.org/10.1016/j.jmb.2009.10.071 [8] Leiman, P.G., Arisaka, F., Van Raaij, M.J., Kostyuchenko, V.A., Aksyuk, A.A., Kanamaru, S. and Rossmann, M.G. (2010) Morphogenesis of the T4 Tail and Tail Fibers. Virology Journal, 7, 355.
http://dx.doi.org/10.1186/1743-422X-7-355 [9] Kanamaru, S., Leiman, P.G., Kostyuchenko, V.A., Chipman, P.R., Mesyanzhinov, V.V., Arisaka, F. and Rossmann, M.G. (2002) Structure of the Cell-Puncturing Device of Bacteriophage T4. Nature, 415, 553-557. http://dx.doi.org/10.1038/415553a [10] Bebeacua, C., Lai, L., Vegge, C.S., Brondsted, L., Van Heel, M., Veesler, D. and Cambillau, C. (2013) Visualizing a Complete Siphoviridae Member by Single-Particle Electron Microscopy: The Structure of Lactococcal Phage TP901-1. Journal of Virology, 87, 1061-1068.
http://dx.doi.org/10.1128/JVI.02836-12 [11] Badasso, M.O., Leiman, P.G., Tao, Y., He, Y., Ohlendorf, D.H., Rossmann, M.G. and Anderson, D. (2000) Purification, Crystallization and Initial X-Ray Analysis of the Head-Tail Connector of Bacteriophage phi29. Acta crystallographica Section D, Biological Crystallography, 56, 1187-1190. http://dx.doi.org/10.1107/S0907444900009239 [12] Tang, L., Marion, W.R., Cingolani, G., Prevelige, P.E. and Johnson, J.E. (2005) Three-Dimensional Structure of the Bacteriophage P22 Tail Machine. The EMBO Journal, 24, 2087-2095. http://dx.doi.org/10.1038/sj.emboj.7600695 [13] Fokine, A., Zhang, Z.H., Kanamaru, S., Bowman, V.D., Aksyuk, A.A., Arisaka, F., Rao, V.B. and Rossmann, M.G. (2013) The Molecular Architecture of the Bacteriophage T4 Neck. Journal of Molecular Biology, 425, 1731-1744. http://dx.doi.org/10.1016/j.jmb.2013.02.012 [14] Bebeacua, C., Tremblay, D., Farenc, C., Chapot-Chartier, M.P., Sadovskaya, I., Van Heel, M., Veesler, D., Moineau, S. and Cambillau, C. (2013) Structure, Adsorption to Host, and Infection Mechanism of Virulent Lactococcal Phage p2. Journal of Virology, 87, 12302-12312.
http://dx.doi.org/10.1128/JVI.02033-13 [15] Xu, J., Hendrix, R.W. and Duda, R.L. (2013) A Balanced Ratio of Proteins from Gene G and Frameshift-Extended Gene GT Is Required for Phage Lambda Tail Assembly. Journal of Molecular Biology, 425, 3476-3487. http://dx.doi.org/10.1016/j.jmb.2013.07.002 [16] Hendrix, R.W. and Johnson, J.E. (2012) Bacteriophage HK97 Capsid Assembly and Maturation. Advances in Experimental Medicine and Biology, 726, 351-363.
http://dx.doi.org/10.1007/978-1-4614-0980-9_15 [17] Arisaka, F. (2005) Assembly and Infection Process of Bacteriophage T4. Chaos, 15, Article ID: 047502. http://dx.doi.org/10.1063/1.2142136 [18] O’Day, K., Schultz, D., Ericsen, W., Rawluk, L. and Howe, M. (1979) Correction and Refinement of the Genetic Map of Bacteriophage Mu. Virology, 93, 320-328.
http://dx.doi.org/10.1016/0042-6822(79)90236-8 [19] Giphart-Gassler, M., Wijffelman, C. and Reeve, J. (1981) Structural Polypeptides and Products of Late Genes of Bacteriophage Mu: Characterization and Functional Aspects. Journal of Molecular Biology, 145, 139-163. http://dx.doi.org/10.1016/0022-2836(81)90338-7 [20] Grundy, F.J. and Howe, M.M. (1985) Morphogenetic Structures Present in Lysates of Amber Mutants of Bacteriophage Mu. Virology, 143, 485-504. http://dx.doi.org/10.1016/0042-6822(85)90388-5 [21] Morgan, G.J., Hatfull, G.F., Casjens, S. and Hendrix, R.W. (2002) Bacteriophage Mu Genome Sequence: Analysis and Comparison with Mu-Like Prophages in Haemophilus, Neisseria and Deinococcus. Journal of Molecular Biology, 317, 337-359. http://dx.doi.org/10.1006/jmbi.2002.5437 [22] Kondou, Y., Kitazawa, D., Takeda, S., Tsuchiya, Y., Yamashita, E., Mizuguchi, M., Kawano, K. and Tsukihara, T. (2005) Structure of the Central Hub of Bacteriophage Mu Baseplate Determined by X-Ray Crystallography of gp44. Journal of Molecular Biology, 352, 976-985.
http://dx.doi.org/10.1016/j.jmb.2005.07.044 [23] Harada, K., Yamashita, E., Nakagawa, A., Miyafusa, T., Tsumoto, K., Ueno, T., Toyama, Y. and Takeda, S. (2013) Crystal Structure of the C-Terminal Domain of Mu Phage Central Spike and Functions of Bound Calcium Ion. Biochimica et Biophysica Acta, 1834, 284-291.
http://dx.doi.org/10.1016/j.bbapap.2012.08.015 [24] Kitazawa, D., Takeda, S., Kageyama, Y., Tomihara, M. and Fukada, H. (2005) Expression and Characterization of a Baseplate Protein for Bacteriophage Mu, gp44. Journal of Biochemistry, 137, 601-606. http://dx.doi.org/10.1093/jb/mvi076 [25] Schuck, P. (2000) Size-Distribution Analysis of Macromolecules by Sedimentation Velocity Ultracentrifugation and Lamm Equation Modeling. Biophysical Journal, 78, 1606-1619.
http://dx.doi.org/10.1016/S0006-3495(00)76713-0 [26] Schuck, P., Perugini, M.A., Gonzales, N.R., Howlett, G.J. and Schubert, D. (2002) Size-Distribution Analysis of Proteins by Analytical Ultracentrifugation: Strategies and Application to Model Systems. Biophysical Journal, 82, 1096- 1111. http://dx.doi.org/10.1016/S0006-3495(02)75469-6 [27] Lebowitz, J., Lewis, M.S. and Schuck, P. (2002) Modern Analytical Ultracentrifugation in Protein Science: A Tutorial Review. Protein Science, 11, 2067-2079. http://dx.doi.org/10.1110/ps.0207702 [28] Suzuki, H., Yamada, S., Toyama, Y. and Takeda, S. (2010) The C-Terminal Domain Is Sufficient for Host-Binding Activity of the Mu Phage Tail-Spike Protein. Biochimica et Biophysica Acta, 1804, 1738-1742. http://dx.doi.org/10.1016/j.bbapap.2010.05.003 [29] Piuri, M. and Hatfull, G.F. (2006) A Peptidoglycan Hydrolase Motif within the Mycobacteriophage TM4 Tape Measure Protein Promotes Efficient Infection of Stationary Phase Cells. Molecular Microbiology, 62, 1569-1585. http://dx.doi.org/10.1111/j.1365-2958.2006.05473.x [30] Rodriguez-Rubio, L., Gutierrez, D., Martinez, B., Rodriguez, A., Gotz, F. and Garcia, P. (2012) The Tape Measure Protein of the Staphylococcus aureus Bacteriophage vB_SauS-phiIPLA35 Has an Active Muramidase Domain. Applied and Environmental Microbiology, 78, 6369-6371.
http://dx.doi.org/10.1128/AEM.01236-12