Identification of a Pseudomonas aeruginosa PAO1 DNA Methyltransferase, Its Targets, and Physiological Roles.

Sebastian Doberenz,Vanessa Jensen,Jonas Korlach,Cathrin Spröer,Olga Reichert,Susanne Häussler,Adrian Kordes,Emanuela Frangipani,Volkhard Kaever,Khai Luong,Denitsa Eckweiler,Boyke Bunk,Jörg Overmann,Stephan Heeb,Pascale F Cossart

doi:10.1128/mbio.02312-16

Abstract

ABSTRACTDNA 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 pathogen Pseudomonas aeruginosa PAO1. 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 affected P. aeruginosa pathogenicity in a Galleria mellonella infection 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 in P. aeruginosa. Since the DNA methylation enzymes are not encoded in the core genome, our findings illustrate how the acquisition of accessory genes can shape the global P. aeruginosa transcriptome and thus may facilitate adaptation to new and challenging habitats.

Highlights

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
We identified an adenosine DNA methyltransferase in the opportunistic pathogen Pseudomonas aeruginosa PAO1, which is responsible for DNA methylation of a conserved sequence motif
We have used single-molecule real-time (SMRT) sequencing to uncover methylated adenines throughout the P. aeruginosa PAO1 genome, and methylation was found to be dependent on the activity of the hsdM DNA MTase encoded by PA2735

Summary

Introduction

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. We used single-molecule real-time (SMRT) sequencing to uncover the genome-wide DNA methylation pattern in the opportunistic pathogen Pseudomonas aeruginosa PAO1. We identified an adenosine DNA methyltransferase in the opportunistic pathogen Pseudomonas aeruginosa PAO1, which is responsible for DNA methylation of a conserved sequence motif. Genomic analysis revealed multiple putative MTases within individual P. aeruginosa genomes, those genes form part of the accessory genome [31] This suggests that methylation is commonly observed in the species P. aeruginosa, the genome-wide methylation patterns are strain specific and depend on the acquisition of specific MTases and their respective specificity domain subunit. SMRT technology represents a powerful tool for characterization of the role of DNA methylation in a wide variety of bacteria and has paved the way for studies on the potential regulatory roles of adenine methylation [33, 36]

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Results

Conclusion