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Journal of Bacteriology, February 2003, p. 1326-1337, Vol. 185, No. 4
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.4.1326-1337.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Effects of the Chromosome Partitioning Protein Spo0J (ParB) on oriC Positioning and Replication Initiation in Bacillus subtilis

Philina S. Lee,1 Daniel Chi-Hong Lin,1,{dagger} Shigeki Moriya,2,{ddagger} and Alan D. Grossman1*

Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139,1 Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma Nara 630-0101, Japan2

Received 3 October 2002/ Accepted 26 November 2002

Spo0J (ParB) of Bacillus subtilis is a DNA-binding protein that belongs to a conserved family of proteins required for efficient plasmid and chromosome partitioning in many bacterial species. We found that Spo0J contributes to the positioning of the chromosomal oriC region, but probably not by recruiting the origin regions to specific subcellular locations. In wild-type cells during exponential growth, duplicated origin regions were generally positioned around the cell quarters. In a spo0J null mutant, sister origin regions were often closer together, nearer to midcell. We found, by using a Spo0J-green fluorescent protein [GFP] fusion, that the subcellular location of Spo0J was a consequence of the chromosomal positions of the Spo0J binding sites. When an array of binding sites (parS sites) were inserted at various chromosomal locations in the absence of six of the eight known parS sites, Spo0J-GFP was no longer found predominantly at the cell quarters, indicating that Spo0J is not sufficient to recruit chromosomal parS sites to the cell quarters. spo0J also affected chromosome positioning during sporulation. A spo0J null mutant showed an increase in the number of cells with some origin-distal regions located in the forespore. In addition, a spo0J null mutation caused an increase in the number of foci per cell of LacI-GFP bound to arrays of lac operators inserted in various positions in the chromosome, including the origin region, an increase in the DNA-protein ratio, and an increase in origins per cell, as determined by flow cytometry. These results indicate that the spo0J mutant produced a significant proportion of cells with increased chromosome content, probably due to increased and asynchronous initiation of DNA replication.

* Corresponding author. Mailing address: Department of Biology, Building 68-530, Massachusetts Institute of Technology, Cambridge, MA 02139. Phone: (617) 253-1515. Fax: (617) 253-2643. E-mail: adg{at}mit.edu.

{dagger} Present address: Tularik, Inc., South San Francisco, CA 94080.

{ddagger} Present address: School of Molecular and Microbial Biosciences, University of Sydney, Sydney, NSW 2006, Australia.

Journal of Bacteriology, February 2003, p. 1326-1337, Vol. 185, No. 4
0021-9193/03/$08.00+0     DOI: 10.1128/JB.185.4.1326-1337.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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