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CcrM, an adenine DNA methyltransferase, is essential for viability in Caulobacter crescentus. The CcrM protein is present only in the predivisional stage of the cell cycle, resulting in cell-cycle-dependent variation of the DNA methylation state of the chromosome. The availability of CcrM is controlled in two ways: (1) the ccrM gene is transcribed only in the predivisional cell, and (2) the CcrM protein is rapidly degraded prior to cell division. We demonstrate here that CcrM is an important target of the Lon protease pathway in C. crescentus. In a lon null mutant, ccrM transcription is still temporally regulated, but the CcrM protein is present throughout the cell cycle because of a dramatic increase in its stability that results in a fully methylated chromosome throughout the cell cycle. Because the Lon protease is present throughout the cell cycle, it is likely that the level of CcrM in the cell is controlled by a dynamic balance between temporally varied transcription and constitutive degradation. We have shown previously that restriction of CcrM to the C. crescentus predivisional cell is essential for normal morphogenesis and progression through the cell cycle. Comparison of the lon null mutant strain with a strain whose DNA remains fully methylated as a result of constitutive expression of ccrM suggests that the effect of Lon on DNA methylation contributes to several developmental defects observed in the lon mutant. These defects include a frequent failure to complete cell division and loss of precise cell-cycle control of initiation of DNA replication. Other developmental abnormalities exhibited by the lon null mutant, such as the formation of abnormally long stalks, appear to be unrelated to altered chromosome methylation state. The Lon protease thus exhibits pleiotropic effects in C. crescentus growth and development.

The initiation of DNA replication is under differential control in Caulobacter crescentus. Following cell division, only the chromosome in the progeny stalked cell is able to initiate DNA replication, while the chromosome in the progeny swarmer cell does not replicate until later in the cell cycle. We have isolated the dnaA gene in order to determine whether this essential and ubiquitous replication initiation protein also contributes to differential replication control in C. crescentus. Analysis of the cloned C. crescentus dnaA gene has shown that the deduced amino acid sequence can encode a 486-amino-acid protein that is 37% identical to the DnaA protein of Escherichia coli. The gene is located 2 kb from the origin of replication. Primer extension analysis revealed a single transcript originating from a sigma 70-type promoter. Immunoprecipitation of a DnaA'-beta-lactamase fusion protein showed that although expression occurs throughout the cell cycle, there is a doubling in the rate of expression just prior to the initiation of replication.

The expression of the Caulobacter ccrM gene and the activity of its product, the M.Ccr II DNA methyltransferase, are limited to a discrete portion of the cell cycle (G. Zweiger, G. Marczynski, and L. Shapiro, J. Mol. Biol. 235:472-485, 1994). Temporal control of DNA methylation has been shown to be critical for normal development in the dimorphic Caulobacter life cycle. To understand the mechanism by which ccrM expression is regulated during the cell cycle, we have identified and characterized the ccrM promoter region. We have found that it belongs to an unusual promoter family used by several Caulobacter class II flagellar genes. The expression of these class II genes initiates assembly of the flagellum just prior to activation of the ccrM promoter in the predivisional cell. Mutational analysis of two M.Ccr II methylation sites located 3 to the ccrM promoter suggests that methylation might influence the temporally controlled inactivation of ccrM transcription. An additional parallel between the ccrM and class II flagellar promoters is that their transcription responds to a cell cycle DNA replication checkpoint. We propose that a common regulatory system coordinates the expression of functionally diverse genes during the Caulobacter cell cycle.Site-specific methylation of chromosomal DNA has been observed in a wide range of organisms, from bacteria to plants and humans. DNA methylation can have a critical role in the regulation of protein-DNA transactions involved in numerous cellular processes, including transcription (5, 8, 28), repair of mutational lesions (18, 25), and transposition (2, 35). The methylation state of the origin of replication is an important factor regulating the initiation of chromosomal replication in Escherichia coli (29). Analysis of the M.Ccr II DNA methyltransferase (MTase) in the bacterium Caulobacter crescentus revealed that chromosomal methylation has an important role in growth and development in this organism (47). (The M.Ccr II DNA MTase protein was referred to by Zweiger et al. [47] as CcrM. We have changed the name herein to conform to accepted nomenclature for DNA methyltransferases. There does not appear to be a cognate restriction enzyme for the M.Ccr II recognition site [GAnTC].) To gain insight into the role of DNA methylation in Caulobacter cell differentiation, we have focused in this work on understanding how M.Ccr II activity is controlled and have found that a common regulatory system appears to coordinate expression of M.Ccr II with other cell cycle-regulated events.C. crescentus exhibits a distinctive cell cycle modulated pattern of DNA adenine methylation (47). GAnTC sites become hemimethylated upon passage of the replication fork, and remethylation of the newly synthesized strand in both new chromosomes is delayed until shortly before cell division, when replication is at or near completion. The onset and duration of the hemimethylated state for a particular site is thus dependent on its position on the chromosome relative to the origin of replication. Transcriptio...

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