title: Identification of aPseudomonas aeruginosaPAO1 DNA Methyltransferase, Its Targets, and Physiological Roles.
abstract: DNA methylation is widespread among prokaryotes, and most DNA methylation reactions are catalyzed by adenine DNA methyltransferases, which are part of restriction-modification (R-M) systems. R-M systems are known for their role in the defense against foreign DNA; however, DNA methyltransferases also play functional roles in gene regulation. In this study, we used single-molecule real-time (SMRT) sequencing to uncover the genome-wide DNA methylation pattern in the opportunistic pathogenPseudomonas aeruginosaPAO1. We identified a conserved sequence motif targeted by an adenine methyltransferase of a type I R-M system and quantified the presence of N6-methyladenine using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Changes in the PAO1 methylation status were dependent on growth conditions and affectedP. aeruginosapathogenicity in aGalleria mellonellainfection model. Furthermore, we found that methylated motifs in promoter regions led to shifts in sense and antisense gene expression, emphasizing the role of enzymatic DNA methylation as an epigenetic control of phenotypic traits inP. aeruginosaSince the DNA methylation enzymes are not encoded in the core genome, our findings illustrate how the acquisition of accessory genes can shape the globalP. aeruginosatranscriptome and thus may facilitate adaptation to new and challenging habitats.IMPORTANCEWith the introduction of advanced technologies, epigenetic regulation by DNA methyltransferases in bacteria has become a subject of intense studies. Here we identified an adenosine DNA methyltransferase in the opportunistic pathogenPseudomonas aeruginosaPAO1, which is responsible for DNA methylation of a conserved sequence motif. The methylation level of all target sequences throughout the PAO1 genome was approximated to be in the range of 65 to 85% and was dependent on growth conditions. Inactivation of the methyltransferase revealed an attenuated-virulence phenotype in theGalleria mellonellainfection model. Furthermore, differential expression of more than 90 genes was detected, including the small regulatory RNAprrF1, which contributes to a global iron-sparing response via the repression of a set of gene targets. Our finding of a methylation-dependent repression of the antisense transcript of theprrF1small regulatory RNA significantly expands our understanding of the regulatory mechanisms underlying active DNA methylation in bacteria.
authors.affiliation: Institute for Molecular Bacteriology, Twincore GmbH, Center for Clinical and Experimental Infection Research, Hannover, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Doberenz
authors.affiliation: Department of Molecular Bacteriology, Helmholtz Centre for Infection Research, Braunschweig, Germany.
authors.forename: Denitsa
authors.initials: D
authors.lastname: Eckweiler
authors.affiliation: Institute for Molecular Bacteriology, Twincore GmbH, Center for Clinical and Experimental Infection Research, Hannover, Germany.
authors.forename: Olga
authors.initials: O
authors.lastname: Reichert
authors.affiliation: Institute for Molecular Bacteriology, Twincore GmbH, Center for Clinical and Experimental Infection Research, Hannover, Germany.
authors.forename: Vanessa
authors.initials: V
authors.lastname: Jensen
authors.affiliation: Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany.
authors.forename: Boyke
authors.initials: B
authors.lastname: Bunk
authors.affiliation: Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany.
authors.forename: Cathrin
authors.initials: C
authors.lastname: Spröer
authors.affiliation: Institute for Molecular Bacteriology, Twincore GmbH, Center for Clinical and Experimental Infection Research, Hannover, Germany.
authors.forename: Adrian
authors.initials: A
authors.lastname: Kordes
authors.affiliation: School of Life Sciences, Centre for Biomolecular Sciences, University of Nottingham, Nottingham, United Kingdom.
authors.forename: Emanuela
authors.initials: E
authors.lastname: Frangipani
authors.affiliation: Pacific Biosciences, Menlo Park, California, USA.
authors.forename: Khai
authors.initials: K
authors.lastname: Luong
authors.affiliation: Pacific Biosciences, Menlo Park, California, USA.
authors.forename: Jonas
authors.initials: J
authors.lastname: Korlach
authors.affiliation: School of Life Sciences, Centre for Biomolecular Sciences, University of Nottingham, Nottingham, United Kingdom.
authors.forename: Stephan
authors.initials: S
authors.lastname: Heeb
authors.affiliation: Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany.
authors.forename: Jörg
authors.initials: J
authors.lastname: Overmann
authors.affiliation: Research Core Unit Metabolomics, Hannover Medical School, Hannover, Germany.
authors.forename: Volkhard
authors.initials: V
authors.lastname: Kaever
authors.affiliation: Institute for Molecular Bacteriology, Twincore GmbH, Center for Clinical and Experimental Infection Research, Hannover, Germany susanne.haeussler@helmholtz-hzi.de.|Department of Molecular Bacteriology, Helmholtz Centre for Infection Research, Braunschweig, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Häussler
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives* / Q000032:analysis; D000818:Animals; D002853:Chromatography, Liquid; D019175:DNA Methylation; D004195:Disease Models, Animal; D044127:Epigenesis, Genetic; D015964:Gene Expression Regulation, Bacterial; D007915:Lepidoptera / Q000382:microbiology; D013058:Mass Spectrometry; D011401:Promoter Regions, Genetic; D011552:Pseudomonas Infections / Q000382:microbiology; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000378:metabolism*; D014774:Virulence
chemical_list: D000225:Adenine; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); C000730567:6-methyladenine