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      DNA Binding of the Cell Cycle Transcriptional Regulator GcrA Depends on N6-Adenosine Methylation in Caulobacter crescentus and Other Alphaproteobacteria

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          Several regulators are involved in the control of cell cycle progression in the bacterial model system Caulobacter crescentus, which divides asymmetrically into a vegetative G1-phase (swarmer) cell and a replicative S-phase (stalked) cell. Here we report a novel functional interaction between the enigmatic cell cycle regulator GcrA and the N6-adenosine methyltransferase CcrM, both highly conserved proteins among Alphaproteobacteria, that are activated early and at the end of S-phase, respectively. As no direct biochemical and regulatory relationship between GcrA and CcrM were known, we used a combination of ChIP (chromatin-immunoprecipitation), biochemical and biophysical experimentation, and genetics to show that GcrA is a dimeric DNA–binding protein that preferentially targets promoters harbouring CcrM methylation sites. After tracing CcrM-dependent N6-methyl-adenosine promoter marks at a genome-wide scale, we show that these marks recruit GcrA in vitro and in vivo. Moreover, we found that, in the presence of a methylated target, GcrA recruits the RNA polymerase to the promoter, consistent with its role in transcriptional activation. Since methylation-dependent DNA binding is also observed with GcrA orthologs from other Alphaproteobacteria, we conclude that GcrA is the founding member of a new and conserved class of transcriptional regulators that function as molecular effectors of a methylation-dependent (non-heritable) epigenetic switch that regulates gene expression during the cell cycle.

          Author Summary

          Methylation of genomic DNA at a specific regulatory site can impact a myriad of processes in eukaryotic cells. In bacteria, methylation at the N6 position of adenosine (m6A) is known to mediate a non-adaptive immunity response to protect cells from foreign DNA. While m6A marks are not known to govern expression of cell cycle genes in Gammaproteobacteria, cell cycle transcription in the model alphaproteobacterium Caulobacter crescentus requires the m6A methyltransferase CcrM that introduces m6A marks at GAnTC sequences and the enigmatic factor GcrA. Investigating if a functional and biochemical relationship exists between CcrM and GcrA, we found that CcrM-dependent m6A marks recruit GcrA to the promoters of cell cycle genes in vitro and in vivo and is required for efficient transcription. GcrA interacts with RNA polymerase, explaining how cell cycle transcription is affected. Importantly, m6A-dependent binding is also seen in GcrA orthologs, indicating that this transcriptional regulatory mechanism by CcrM and GcrA is conserved in Alphaproteobacteria.

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          Most cited references 24

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          SOPMA: significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments.

          Recently a new method called the self-optimized prediction method (SOPM) has been described to improve the success rate in the prediction of the secondary structure of proteins. In this paper we report improvements brought about by predicting all the sequences of a set of aligned proteins belonging to the same family. This improved SOPM method (SOPMA) correctly predicts 69.5% of amino acids for a three-state description of the secondary structure (alpha-helix, beta-sheet and coil) in a whole database containing 126 chains of non-homologous (less than 25% identity) proteins. Joint prediction with SOPMA and a neural networks method (PHD) correctly predicts 82.2% of residues for 74% of co-predicted amino acids. Predictions are available by Email to or on a Web page (
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            N6-methyl-adenine: an epigenetic signal for DNA-protein interactions.

            N(6)-methyl-adenine is found in the genomes of bacteria, archaea, protists and fungi. Most bacterial DNA adenine methyltransferases are part of restriction-modification systems. Certain groups of Proteobacteria also harbour solitary DNA adenine methyltransferases that provide signals for DNA-protein interactions. In gamma-proteobacteria, Dam methylation regulates chromosome replication, nucleoid segregation, DNA repair, transposition of insertion elements and transcription of specific genes. In Salmonella, Haemophilus, Yersinia and Vibrio species and in pathogenic Escherichia coli, Dam methylation is required for virulence. In alpha-proteobacteria, CcrM methylation regulates the cell cycle in Caulobacter, Rhizobium and Agrobacterium, and has a role in Brucella abortus infection.
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              MipZ, a spatial regulator coordinating chromosome segregation with cell division in Caulobacter.

              Correct positioning of the division plane is a prerequisite for the generation of daughter cells with a normal chromosome complement. Here, we present a mechanism that coordinates assembly and placement of the FtsZ cytokinetic ring with bipolar localization of the newly duplicated chromosomal origins in Caulobacter. After replication of the polarly located origin region, one copy moves rapidly to the opposite end of the cell in an MreB-dependent manner. A previously uncharacterized essential protein, MipZ, forms a complex with the partitioning protein ParB near the origin of replication and localizes with the duplicated origin regions to the cell poles. MipZ directly interferes with FtsZ polymerization, thereby restricting FtsZ ring formation to midcell, the region of lowest MipZ concentration. The cellular localization of MipZ thus serves the dual function of positioning the FtsZ ring and delaying formation of the cell division apparatus until chromosome segregation has initiated.

                Author and article information

                Role: Editor
                PLoS Genet
                PLoS Genet
                PLoS Genetics
                Public Library of Science (San Francisco, USA )
                May 2013
                May 2013
                30 May 2013
                : 9
                : 5
                [1 ]Interdisciplinary Research Institute USR3078, CNRS–Université Lille Nord de France, Villeneuve d'Ascq, France
                [2 ]Department of Microbiology and Molecular Medicine, University of Geneva, Geneva, Switzerland
                [3 ]Laboratoire de Biométrie et Biologie Evolutive UMR5558, CNRS–Université Lyon 1–INRIA, Villeurbanne, France
                [4 ]Laboratoire GEPV UMR 8198, CNRS–Université Lille 1–Université Lille Nord de France, Villeneuve d'Ascq, France
                Universidad de Sevilla, Spain
                Author notes

                PH Viollier is an Associate Editor of PLOS Genetics.

                Conceived and designed the experiments: EG Biondi, PH Viollier. Performed the experiments: A Fioravanti, C Fumeaux, SS Mohapatra, C Bompard. Analyzed the data: M Brilli, A Frandi, C Bompard. Contributed reagents/materials/analysis tools: V Castric, V Villeret. Wrote the paper: EG Biondi, PH Viollier, A Fioravanti, C Fumeaux, M Brilli, A Frandi, C Bompard. Conceived and designed the structural biology experiments: C Bompard.


                Current address: Fondazione Edmund Mach/CRI, Functional Genomics, San Michele all'Adige, Italy


                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Page count
                Pages: 16
                EG Biondi's lab is funded by ANR_11_SVJ3_003_01, the Région Nord Pas-de-Calais, and the CPER-CIA. PH Viollier is funded by SNF grant 31003A_143660 and HFSP RGP0051/2010. A Fioravanti's PhD fellowship is funded by University of Lille1 and Region Nord-Pas-de-Calais; SS Mohapatra's postdoctoral fellowship is funded by ANR_11_SVJ3_003_01. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Research Article
                DNA modification
                Gene Expression
                DNA transcription
                Gene Networks
                Model Organisms
                Prokaryotic Models
                Caulobacter Crescentus



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