Identification of a Pseudomonas aeruginosa PAO1 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 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.

Sources of artifact in measurements of 6mA and 4mC abundance in eukaryotic genomic DNA

“…SMRTseq takes advantage of the effects of base modifications in the DNA template on the kinetics of incorporation of complementary nucleotides, to infer DNA methylation events [28]. This technique has been validated in a number of unicellular species [30,31,[43][44][45][46] and we and others, used SMRTseq to map 6mA events in metazoa [10,13,15,20,30,47]. SMRTseq is appealing because it provides an independent method for validating the presence of 6mA and can identify modified bases in their sequence context at nucleotide resolution.…”

Genome sequence of Pseudomonas aeruginosa PAO1161, a PAO1 derivative with the ICEPae1161 integrative and conjugative element

“…1a). Interestingly, two mutations identified in PAO1161 in comparison to PAO1-UW, that resulted in an introduction of premature stop codons mapped to PA2735 gene, recently shown to encode a N6adenosine DNA methyltransferase acting on a conserved sequence GATC(N) 6 GTC [50,51], and PA1939, encoding a putative overcoming lysogenization defect Table 1 SNPs and indels identified in P. aeruginosa PAO1161 genome, resulting in expression of truncated proteins. The effect of a mutation is predicted using the PAO1-UW genome as a reference.…”

On-Demand Micro-Power Generation from an Origami-Inspired Paper Biobattery Stack

“…We previously demonstrated that the wild-type Pseudomonas aeruginosa PAO1 can be freeze-dried and rehydrated so that their metabolism generates power (Figure 5c) [29]. Furthermore, our recent literature survey indicates that P. aeruginosa can produce protecting proteins under oxidative stress such as freezedrying (Figure 5b) [37,38], which will probably improve their survival rate during the process. The rehydration of freeze-dried exoelectrogens is a critical step for on-demand power generation [39].…”