DNA methylation; DNA mismatch repair (MMR); Very short patch (VSP) repair; Transcription; Epigenetics; Bacteria; Cell cycle control
Woldemeskel Selamawit Abi, Daitch Allison K., Alvarez Laura, Panis Gaël, Zeinert Rilee, Gonzalez Diego, Smith Erika, Collier Justine, Chien Peter, Cava Felipe, Viollier Patrick H., Goley Erin D. (2020), The conserved transcriptional regulator CdnL is required for metabolic homeostasis and morphogenesis in Caulobacter, in PLOS Genetics
, 16(1), e1008591-e1008591.
CollierJustine, FrandiAntonio (2019), Multilayered control of chromosome replication in Caulobacter crescentus., in Biochemocal Society Transactions
Mouammine Annabelle, Collier Justine (2018), The impact of DNA methylation in Alphaproteobacteria, in Molecular Microbiology
, 110(1), 1-10.
MouammineAnnabelle, EichKatharina, FrandiAntonio, CollierJustine (2018), Control of proline utilization by the Lrp-like regulator PutR in Caulobacter crescentus., in Scientific Reports
Collier Justine (2018), Cell division control in Caulobacter crescentus, in Biochim. Biophys. Acta (BBA) Gene Regulatory Mechanisms
, (17), 30408.
Methylation is a DNA modification frequently found in all kingdoms of life. Epigenetics has been mostly studied in eukaryotes such as fungi, plants and humans, where it has now become evident that it plays important roles during embryogenesis, cellular differentiation, genomic imprinting and cancer development. Methylated cytosines (m5C and m4C) and adenines (m6A) are also frequently found in bacterial genomes due to the activity of restriction-modification systems acting as immigration controllers preventing horizontal gene transfers, but also of orphan DNA methyl-transferases (MTases) involved in DNA mismatch repair and/or in regulating gene expression to control a variety of processes including pathogenesis and virulence. Our previous work on the cell cycle-regulated DNA MTase CcrM that is found in most Alphaproteobacteria, has uncovered a tight connection between adenine methylation and cell cycle control in the model C. crescentus bacterium. Still, too little is known on which genes are directly regulated by DNA methylation in Alphaproteobacteria and on epigenetic regulators that can sense DNA methylation marks to modulate gene expression. The first aim of this proposal is to address this issue directly. We will use (epi)genomics and classical molecular biology methods to identify genes regulated by DNA methylation in the environmental bacterium Caulobacter crescentus and in the distantly-related Agrobacterium tumefaciens plant pathogen. We will then select the most interesting genes, preferentially regulated in a CcrM-dependent manner in all three bacteria, to identify novel and conserved epigenetic regulators in C. crescentus using forward genetics or biochemical screens. Considering that the expression of the majority of the genes suspected to be regulated by DNA methylation is cell cycle-regulated and/or encoding proteins involved in DNA metabolism in C. crescentus, we hope to uncover novel cell cycle regulators that we will characterize in more details to reveal their mechanism(s) of action and their function(s) in Alphaproteobacteria.Although DNA methylation plays important roles in so many organisms, it comes with a serious drawback: m5C can be accidentally deaminated into thymines (T). Resulting TG DNA mismatches must be corrected before they get replicated or they can turn into permanent mutations. Bacteria use Vsr endonucleases during the Very Small Patch (VSP) repair process to eliminate these mismatches but the regulation and the specificity of this process remain unclear, especially in bacteria that have multiple cytosine MTases and multiple Vsr-like proteins like C. crescentus. The second aim of this proposal is to characterize and visualize the VSP process in live C. crescentus cells using molecular genetics and state-of-the-art microscopy experiments. Considering that VSP repair also requires MutS and MutL, two proteins also involved in the repair of DNA mismatches escaping from the proofreading activity of the replicative DNA polymerase (MMR), we will study the potential competition or cooperation between MMR and VSP repair in C. crescentus cells. Defects in DNA mismatch repair are a concern for human health as they can promote the incidence of antibiotic-resistant bacterial strains or of several cancers and neurodegenerative diseases.