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Journal of Bacteriology, August 2009, p. 4807-4814, Vol. 191, No. 15
0021-9193/09/$08.00+0     doi:10.1128/JB.00435-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Replication Initiator DnaA of Escherichia coli Changes Its Assembly Form on the Replication Origin during the Cell Cycle{triangledown}

Shingo Nozaki,1 Hironori Niki,2 and Tohru Ogawa1*

Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan,1 Microbial Genetics Laboratory, Genetic Strains Research Center, National Institute of Genetics, Yata 1111, Mishima 411-8540, Japan2

Received 31 March 2009/ Accepted 27 May 2009

DnaA is a replication initiator protein that is conserved among bacteria. It plays a central role in the initiation of DNA replication. In order to monitor its behavior in living Escherichia coli cells, a nonessential portion of the protein was replaced by a fluorescent protein. Such a strain grew normally, and flow cytometry data suggested that the chimeric protein has no substantial loss of the initiator activity. The initiator was distributed all over the nucleoid. Furthermore, a majority of the cells exhibited certain distinct foci that emitted bright fluorescence. These foci colocalized with the replication origin (oriC) region and were brightest during the period spanning the initiation event. In cells that had undergone the initiation, the foci were enriched in less intense ones. In addition, a significant portion of the oriC regions at this cell cycle stage had no colocalized DnaA-enhanced yellow fluorescent protein (EYFP) focus point. It was difficult to distinguish the initiator titration locus (datA) from the oriC region. However, involvement of datA in the initiation control was suggested from the observation that, in {Delta}datA cells, DnaA-EYFP maximally colocalized with the oriC region earlier in the cell cycle than it did in wild-type cells and oriC concentration was increased.

* Corresponding author. Mailing address: Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan. Phone: 81-52-789-2584. Fax: 81-52-789-5732. E-mail: t.ogawa{at}nagoya-u.jp

{triangledown} Published ahead of print on 5 June 2009.

Journal of Bacteriology, August 2009, p. 4807-4814, Vol. 191, No. 15
0021-9193/09/$08.00+0     doi:10.1128/JB.00435-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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