DNA methylation in Caulobacter and other Alphaproteobacteria during cell cycle progression Saswat S. Mohapatra, Antonella Fioravanti, and Emanuele G. Biondi Interdisciplinary Research Institute, CNRS–Universite ´ Lille Nord de France, 50 Avenue Halley, Villeneuve d’Ascq, France In Caulobacter crescentus, methylation of DNA by CcrM plays an important part in the regulation of cell cycle progression. Thanks to this methyltransferase, the ac- tivity of which is cell cycle regulated, the chromosome transitions between a hemimethylated state in the S- phase to a fully methylated condition in the G1 and G2 phases. Any perturbation in CcrM expression, such as depletion or constitutive expression, causes severe de- velopmental defects. Several studies suggest that the role of CcrM is conserved across the Alphaproteobac- teria. In the past few years, the importance of methyl- ation on the expression of cell cycle regulated genes has emerged, suggesting that CcrM-dependent methylation can direct the binding of transcription factors to specific methylated sequences and affect the expression of genes depending on the methylation state of their pro- moters. CcrM activity has recently been linked to GcrA, a cell cycle master regulator that controls the expression of several genes during S-phase. Here, we review recent findings that establish the global role of methylation in cell cycle progression, and also explore the significance of a CcrM–GcrA epigenetic module that has co-evolved in Alphaproteobacteria, including Caulobacter, in con- trolling several genes involved in cell division, polarity, and motility. Cell cycle progression in Caulobacter DNA, the molecule that stores the information of living organisms, can be modified by methylation, resulting in an epigenetic control of the genetic program. In bacteria, this epigenetic modification is involved in several important physiological processes, such as in the restriction-modifi- cation systems, control of DNA replication, DNA mis- match repair, virulence mechanisms (Box 1), and the cell cycle. Caulobacter crescentus (further referred as Caulobacter) is a remarkable model system for the investigation of cell cycle progression and generation of cellular asymmetry in bacteria [1]. Recent discoveries have helped to elucidate the role of methylation by the methyltransferase CcrM [2,3] as an important regulatory mechanism controlling cell cycle progression in Caulobacter and possibly other Alphaproteobacteria [4–6]. Although the role of methyla- tion during the cell cycle progression has been reviewed before [7], here we will introduce several recent studies that shed light on the global role of CcrM-methylation as a transcriptional modulator, and more significantly we will explain the link between CcrM methylation and the tran- scriptional regulator GcrA as a new epigenetic switch controlling gene expression [8,9]. At every cell division, Caulobacter replicates only once the single circular chromosome and produces two cell types that are morphologically and functionally different (Figure 1). The stalked cell, which is capable of replicating the chromosome and producing new cells [10], possesses a polar tubular foot (the stalk) made by an extension of the cell envelope [11]. The newly formed cells, named swarmer cells, are generated by the stalked cell at the opposite pole of the stalk, and they are in a vegetative state [12]. Swarmer cells are motile, swimming using a single polar flagellum, and possess polar pili. Through the integration of environmental signals, the swarmer cell differentiates into a stalked cell by losing the flagellar appendix and pili and building a stalk at the same polar location. Next, the newly formed stalked cell initiates the replication of the chromosome. The predivisional cell experiences the com- plex coordination of at least five major cell cycle functions: cellular growth, DNA replication, chromosome segrega- tion, biogenesis of polar structures, and cell division. This remarkable asymmetrical cellular development, rare in other bacterial groups, is a distinct characteristic of the Alphaproteobacteria that includes Caulobacter [13–15]. Although most of the key elements of the regulatory circuit of the Caulobacter cell cycle are now known (Box 2), the full comprehension of this complex phenomenon is probably far from being achieved. In Caulobacter, the DNA methyltransferase CcrM methylates both adenosines of the palindromic GAnTC sites in the upper or lower strands [3]. CcrM is not associ- ated with a restriction enzyme and thus it is defined as an orphan methyltransferase. CcrM is active only for a short period at the end of S-phase in the late predivisional cells. Therefore, until the start of a new round of DNA replica- tion, the chromosome is fully methylated in both strands, Review 0966-842X/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tim.2014.05.003 Corresponding author: Biondi, E.G. (emanuele.biondi@iri.univ-lille1.fr). Keywords: Caulobacter crescentus; Alphaproteobacteria; CcrM; GcrA; bacterial cell cycle; N6 adenosine methylation. TIMI-1097; No. of Pages 8 Trends in Microbiology xx (2014) 1–8 1