DNA Methyltransferase M.PaeTBCFI in a chronically adapted Pseudomonas aeruginosa strain

Pseudomonas aeruginosa is an opportunistic pathogen which causes acute and chronic infections that are difficult to treat. Comparative genomic analysis has showed a great genome diversity among P. aeruginosa clinical strains and revealed important regulatory traits during chronic adaptation. While current investigation of epigenetics of P. aeruginosa is still lacking, understanding the epigenetic regulation may provide biomarkers for diagnosis and reveal important regulatory mechanisms. The present study focused on characterization of DNA methyltransferases (MTases) in a chronically adapted P. aeruginosa clinical strain TBCF10839. Single-molecule real-time sequencing (SMRT-seq) was used to characterize the methylome of TBCF. RCCANNNNNNNTGAR and TRGANNNNNNTGC were identified as target motifs of DNA MTases, M.PaeTBCFI and M.PaeTBCFII, respectively. P. aeruginosa TBCF were grown in Luria-Bertani (LB) liquid media at 37°C with constant shaking at 220 rpm with aeration. The in-frame deletion mutagenesis of methyltransferase ap was performed through two-step allelic exchange. P. aeruginosa TBCF and mutants were cultured to mid log phase. Total RNA extraction and purification was performed for 3 biological replicates of each strain using RNeasy Mini Kit (QIAGEN) and RNase-Free DNase Set (QIAGEN) according to the manufacturer's protocol. 1ug of total RNA was used for the construction of sequencing libraries. RNA sequencing was performed on Illumina NovaSeq 6000 sequencing platform generating 150 bp paired-end reads. Raw reads of the samples were preprocessed and analyzed using RSEM v1.3.1 with unique mapping setting (--bowtie-m), using P. aeruginosa TBCF genome as reference.