Isolation, Purification and Characterization of Nucleoids from Synechococcus elongatus PCC 7942


The genomic DNA of bacteria is highly compacted in one or a few bodies known as nucleoids. In order to understand the overall configuration and physiological activities of the cyanobacterial nucleoid under various growth conditions and the role(s) of each nucleoid protein in clock function, thylakoid membrane-associated nucleoids from the Synechococcus elongatus (se) PCC 7942 strain were isolated and purified in presence of spermidine at low salt concentrations by sucrose density gradient centrifugation. The sedimentation rates, protein/DNA composition and microscopic appearances as well as variation in structural components of clock proteins from the isolated nucleoids were compared under identical conditions. Microscopic appearances of the nucleoids were consistent with the sedimentation profiles. The nucleoid structure in the wild type was more tightly compacted than that in the KaiABC mutant strain. Western immunoblot analyses revealed that the KaiC was associated with the nucleoid fraction whereas maximum KaiA was localized in the cytosolic fraction, supposedly in association with the translation machinery.

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Talukder, A. and Kondo, T. (2014) Isolation, Purification and Characterization of Nucleoids from Synechococcus elongatus PCC 7942. Advances in Microbiology, 4, 1105-1116. doi: 10.4236/aim.2014.415121.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Hecht, R.M., Taggart, R.T. and Pettijohn, D.E. (1975) Size and DNA Content of Purified E. coli Nucleoids Observed by Fluorescence Microscopy. Nature, 253, 60-62.
[2] Worcel, A. and Burgi, E. (1972) On the Structure of the Folded Chromosome of Escherichia coli. Journal of Molecular Biology, 71, 127-147.
[3] Pettijohn, D.E. (1982) Structure and Properties of the Bacterial Nucleoid. Cell, 30, 667-669.
[4] Woldringh, C.L. and Nanninga, N. (1985) Structure of the Nucleoid and Cytoplasm in the Intact Cell. In: Nenninga, N., Ed., Molecular Cytology of Escherichia coli, Academic Press, London, 161-197.
[5] Schmid, M.B. (1990) More than Just “Histone-Like” Proteins. Cell, 63, 451-453.
[6] Pettijohn, D.E. (1966) The Nucleoid. In: Neidhardt, F.C., Ed., Escherichia coli and Salmonella, American Society for Microbiology, Washington DC, 158-166.
[7] Flashner,Y. and Gralla, J.D. (1988) DNA Dynamic Flexibility and Protein Recognition-Differential Stimulation by Bacterial Histone-Like Protein HU. Cell, 54, 713-721.
[8] Finkel, S.E. and Johnson, R.C. (1992) The Fis Protein: It’s Not Just for DNA Inversion Anymore. Molecular Microbiology, 6, 3257-3265.
[9] Ishihama, A. (1999) Modulation of the Nucleoid, the Transcription Apparatus and the Translation Machinery for Stationary Phase Survival. Genes to Cells, 4, 135-143.
[10] Ptacin, J.L. and Shapiro, L. (2013) Chromosome Architecture Is a Key Element of Bacterial Cellular Organization. Cell Microbiology, 15, 45-52.
[11] Rouviere-Yaniv, J., Yaniv, M. and Germond, J.E. (1979) Escherichia coli DNA-Binding Protein HU Forms Nucleosome-Like Structure with Circular Double-Stranded DNA. Cell, 17, 265-274.
[12] Drlica, K. and Rouviere-Yaniv, J. (1987) Histone-Like Proteins of Bacteria. Microbial Review, 51, 301-319.
[13] Dürrenberger, M., Bjornsti, M.A., Uetz, T., Hobot, J.A. and Kellenberger, E. (1988) Intracellular Localization of the Histone Like Protein HU in Escherichia coli. Journal of Bacteriology, 170, 4757-4768.
[14] Pettijohn, D.E. (1988) Histone-Like Proteins and Bacterial Chromosome Structure. Journal of Biological Chemistry, 263, 12793-12796.
[15] Ball, C.A., Osuna, R., Ferguson, K.C. and Johnson, R.C. (1992) Dramatic Changes in Fis Levels upon Nutrient Upshift in Escherichia coli. Journal of Bacteriology, 174, 8043-8056.
[16] Almirón, M., Link, A.J., Furlong, D. and Kolter, R. (1992) A Novel DNA Binding Protein with Regulatory and Protective Roles in Starved Escherichia coli. Genes and Development, 6, 2646-2654.
[17] Ditto, M.D., Roberts, D. and Weisberg, R.A. (1994) Growth Phase Variation of Integration Host Factor level in Escherichia coli. Journal of Bacteriology, 176, 3738-3748.
[18] Broyles, S. and Pettijohn, D. (1986) Interaction of the Escherichia coli HU Protein with DNA. Evidence for Formation of Nucleosome-Like Structures with Altered DNA Helical Pitch. Journal of Molecular Biology, 187, 47-60.
[19] Atlung, T. and Ingmer, H. (1997) H-NS: A Modulator of Environmentally Regulated Gene Expression. Molecular Microbiology, 24, 7-17.
[20] Talukder, A.A. and Ishihama, A. (1999) Twelve Species of the Nucleoid-Protein from Escherichia coli: Sequence Recognition Specificity and DNA-Binding Affinity. Journal of Biological Chemistry, 274, 33105-33113.
[21] Talukder, A.A., Iwata, A., Nishimura, A., Ueda, S. and Ishihama, A. (1999) Growth Phase-Dependent Variation in Protein Composition of the Escherichia coli Nucleoid. Journal of Bacteriology, 181, 6361-6370.
[22] Talukder, A.A., Hiraga, S. and Ishihama, A. (2000) Two Types of Localization of the DNA-Binding Proteins within the Escherichia coli Nucleoid. Genes Cells, 5, 613-626.
[23] Talukder, A.A. (2005) Survival and Death in Bacteria. In: Yamada, M., Ed., Structure and Composition of Escherichia coli Nucleoid, Research Signpost, Kerala, 77-101.
[24] Talukder, A.A., Hossain, M.A., Yamada, M. and Ishihama, A. (2006) Nucleoids Dynamics in Escherichia coli: A Growth Phase Dependent Process. Bangladesh Journal of Microbiology, 23, 81-88.
[25] Dunlap, J.C. (1999) Molecular Basis for Circadian Clocks. Cell, 96, 271-290.
[26] Iwasaki, H., Taniguchi, Y., Ishiura, M. and Kondo, T. (1999) Physical Interactions among Circadian Clock Proteins KaiA, KaiB and KaiC in Cyanobacteria. EMBO Journal, 18, 1137-1145.
[27] Kornberg, T., Lockwood, A. and Worcel, A. (1974) Replication of the Escherichia coli Chromosome with a Soluble Enzyme System. Proceedings of the National Academy of Sciences of the United States of America, 71, 3189-3193.
[28] Murphy, L.D. and Zimmerman, S.B. (1997) Isolation and Characterization of Spermidine Nucleoids from Escherichia coli. Journal of Structural Biology, 199, 336-346.
[29] Jishage, M. and Ishihama, A. (1995) Regulation of RNA Polymerase Sigma Subunit Synthesis in Escherichia coli: Intracellular Levels of Sigma 70 and Sigma 38. Journal of Bacteriology, 177, 6832-6835.
[30] Hiraga, S., Ichinose, C., Niki, H. and Yamazoe, M. (1998) Cell Cycle-Dependent Duplication and Bidirectional Migration of SeqA-Associated DNA-Protein Complexes in E. coli. Molecular Cell, 1, 381-387.
[31] Makinoshima, H., Nishimura, A. and Ishihama, A. (2002) Fractionation of Escherichia coli Cell Populations at Different Stages during Growth Transition to Stationary Phase. Molecular Microbiology, 43, 269-279.
[32] Makinoshima, H., Aizawa, S.I., Hayashi, H., Miki, T., Nishimura, A. and Ishihama, A. (2003) Growth Phase-Coupled Alternations in Cell Structure and Function of Escherichia coli. Journal of Bacteriology, 185, 1338-1345.
[33] Mori, T., Binder, B. and Johnson, C.H. (1996) Circadian Gating of Cell Division in Cyanobacteria Growing with Average Doubling Times of Less than 24 Hours. Proceedings of the National Academy of Sciences of the United States of America, 93, 10183-10188.
[34] Stonington, O.G. and Pettijohn, D.E. (1771) The Folded Genome of Escherichia coli Isolated in a Protein-DNA-RNA Complex. Proceedings of the National Academy of Sciences of the United States of America, 68, 6-9.
[35] Hecht, R.M., Stimpson, D. and Pettijohn, D.E. (1977) Sedimentation Properties of the Bacterial Chromosomes as an Isolated Nucleoid and as an Unfolded DNA Fiber: Chromosomal DNA Folding Measured by Rotor Speed Effects. Journal of Molecular Biology, 111, 257-277.
[36] Hinnebusch, B. and Bendich, A.J. (1997) The Bacterial Nucleoid Visualized by Fluorescence Microscopy of Cells Lysed within Agarose: Comparison of Escherichia coli and Spirochetes of the Genus Borrelia. Journal of Bacteriology, 179, 2228-2237.
[37] Murphy, L.D. and Zimmerman, S.B. (1997) Stabilization of Compact Spermidine Nucleoids from Escherichia coli under Crowded Conditions: Implication for in Vivo Nucleoid Structure. Journal of Structural Biology, 199, 321-335.
[38] Zimmerman, S.B. and Murphy, L.D. (2001) Release of Compact Nucleoids with Characteristic Shapes from Escherichia coli. Journal of Bacteriology, 183, 5041-5049.
[39] Weitao, T., Nordstrõm, K. and Dasgupta, S. (1999) Mutational Suppression of mukB and seqA Phenotypes Might Arise from Their Opposing Influences on the Escherichia coli Nucleoid Structure. Molecular Microbiology, 34, 157-168.
[40] Åkerlurd, T., Nordstrõm, K. and Bernander, R. (1995) Analysis of Cell Size and DNA Content in Exponentially Growing and Stationary-Phase Batch Cultures of Escherichia coli. Journal of Bacteriology, 177, 6791-6797.
[41] Nitta, K., Nagayama, K., Danev, R. and Kaneko, Y. (2009) Visualization of BrdU-Labelled DNA in Cyanobacterial Cells by Hilbert Differential Contrast Transmission Electron Microscopy. Journal of Microscopy, 234, 118-123.
[42] Sato, N. (2001) Was the Evolution of Plastid Genetic Machinery Discontinuous? Trends in Plant Science, 6, 151-155.
[43] Nakahira, Y., Katayama, M., Miyashita, H., Kutsuna, S., Iwasaki, H., Oyama, T. and Kondo, T. (2004) Global Gene Repression by KaiC as a Master Process of Prokaryotic Circadian System. Proceedings of the National Academy of Sciences of the United States of America, 101, 881-885.
[44] Kitayama, Y., Iwasaki, H., Nishiwaki, T. and Kondo, T. (2003) KaiB Functions as an Attenuator of KaiC Phosphorylation in the Cyanobacterial Circadian Clock System. EMBO Journal, 22, 2127-2134.
[45] Sato, N., Nakayama, M. and Hase, T. (2001) The 70-kDa Major DNA-Compacting Protein of the Chloroplast Nucleoid Is Sulfite Reductase. FEBS Letters, 318, 347-350.
[46] Mori, T., Saveliev, S.V., Xu, Y., Stafford, W.F., Cox, M.M., Inman, R.B. and Johnson, C.H. (2002) Circadian Clock Protein KaiC forms ATP-Dependent Hexameric Rings and Binds DNA. Proceedings of the National Academy of Sciences of the United States of America, 99, 17203-17208.
[47] Smith, R.M. and Williams, S.B. (2006) Circadian Rhythms in Gene Transcription Impared by Chromosome Compaction in the Cyanobacterium Synechococcus elongatus. Proceedings of the National Academy of Sciences of the United States of America, 103, 8564-8569.
[48] Kobayashi, T., Takahara, M., Miyagishima, S., Kuroiwa, H., Sasaki, N., Ohta, N., Matsuzaki, M. and Kuroiwa, T. (2002) Detection and Localization of a Chloroplast-Encoded HU-Like Protein That Organizes Chloroplast Nucleoids. The Plant Cell, 14, 1579-1589.
[49] Jeong, S.Y., Rose, A. and Meier, I. (2003) MFP1 Is a Thylakoid-Associated, Nucleoid-Binding Protein with a Coiled-Coil Structure. Nucleic Acids Research, 31, 5175-5185.
[50] Kabeya, Y., Nakanishi, H., Suzuki, K., Ichikawa, T., Kondou, Y., Matsui, M. and Miyagishima, S.Y. (2010) The YlmG Protein Has a Conserved Function Related to the Distribution of Nucleoids in Chloroplasts and Cyanobacteria. BMC Plant Biolology, 10, 57.
[51] Durham, K.A. and Bullerjahn, G.S. (2002) Immunocytochemical Localization of the Stress-Induced DpsA Protein in the Cyanobacterium Synechococcus sp. Strain PCC 7942. Journal of Basic Microbiology, 42, 362-372.<367::AID-JOBM367>3.0.CO;2-T
[52] Zhang, L., Paakkarinen, V., van Wijk, K.L. and Aro, E.M. (1999) Co-Translational Assembly of the D1 Protein into Photosystem II. Journal of Biological Chemistry, 274, 16062-16067.
[53] Rohl, T. and van Wijk, K.L. (2001) In Vitro Reconstitution of Insertion and Processing of Cytochrome f in a Homologous Chloroplast Translation System. Journal of Biological Chemistry, 276, 35465-35472.
[54] Johnson, C.H., Eglim, M. and Stewart, P.L. (2008) Structural Insights into a Circadian Oscillator. Science, 322, 697-701.

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