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Journal of Bacteriology, April 1999, p. 1984-1993, Vol. 181, No. 7
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Chromosome Methylation and Measurement of Faithful, Once and Only Once per Cell Cycle Chromosome Replication in Caulobacter crescentus

Gregory T. Marczynski*

Department of Microbiology & Immunology, McGill University, Montreal, Quebec, Canada H3A 2B4

Received 29 September 1998/Accepted 19 January 1999

Caulobacter crescentus exhibits cell-type-specific control of chromosome replication and DNA methylation. Asymmetric cell division yields a replicating stalked cell and a nonreplicating swarmer cell. The motile swarmer cell must differentiate into a sessile stalked cell in order to replicate and execute asymmetric cell division. This program of cell division implies that chromosome replication initiates in the stalked cell only once per cell cycle. DNA methylation is restricted to the predivisional cell stage, and since DNA synthesis produces an unmethylated nascent strand, late DNA methylation also implies that DNA near the replication origin remains hemimethylated longer than DNA located further away. In this report, both assumptions are tested with an engineered Tn5-based transposon, Tn5Omega -MP. This allows a sensitive Southern blot assay that measures fully methylated, hemimethylated, and unmethylated DNA duplexes. Tn5Omega -MP was placed at 11 sites around the chromosome and it was clearly demonstrated that Tn5Omega -MP DNA near the replication origin remained hemimethylated longer than DNA located further away. One Tn5Omega -MP placed near the replication origin revealed small but detectable amounts of unmethylated duplex DNA in replicating stalked cells. Extra DNA synthesis produces a second unmethylated nascent strand. Therefore, measurement of unmethylated DNA is a critical test of the "once and only once per cell cycle" rule of chromosome replication in C. crescentus. Fewer than 1 in 1,000 stalked cells prematurely initiate a second round of chromosome replication. The implications for very precise negative control of chromosome replication are discussed with respect to the bacterial cell cycle.

* Mailing address: Dept. of Microbiology & Immunology, McGill University, 3775 University St., Montreal, Quebec, Canada H3A 2B4. Phone: (514) 398-3917. Fax: (514) 398-7052. E-mail: GMARCZYNSKI{at}NEXUS.MICROIMM.MCGILL.CA.

Journal of Bacteriology, April 1999, p. 1984-1993, Vol. 181, No. 7
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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