DNA Methyltransferase regulates nitric oxide homeostasis and virulence in a chronically adapted Pseudomonas aeruginosa strain

Opportunistic pathogens such as Pseudomonas aeruginosa adapt their genomes rapidly during chronic infections. Understanding their epigenetic regulation may provide biomarkers for diagnosis and reveal novel regulatory mechanisms. We performed single-molecule real-time sequencing (SMRT-seq) to characterize the methylome of a chronically adapted P. aeruginosa clinical strain TBCF10839. Two N6-methyl-adenine (6mA) methylation recognition motifs (RCCANNNNNNNTGAR and TRGANNNNNNTGC) were identified and predicted as new type I methylation sites using REBASE analysis. We confirmed that motif TRGANNNNNNTGCwas methylated by MTase M.PaeTBCFII, according to methylation sensitivity assays in vivo and vitro. Transcriptomic analysis showed that ΔM.PaeTBCFII knockout mutant significantly downregulated nitric oxide reductase (NOR) regulating and coding gene expression such as nosR and norB, which contain methylated motifs in their promoters or coding regions. ΔM.PaeTBCFII exhibited reduced intercellular survival capacity in NO-producing RAW 264.7 macrophages and attenuated virulence in Galleria mellonella infection model; the complemented strain recovered these defective phenotypes. Further phylogenetic analysis demonstrated that homologs of M.PaeTBCFII occur frequently in P. aeruginosa sp as well as other bacterial species. Our work therefore provided new insights on the relationship between DNA methylation, NO detoxification, and bacterial virulence, laying a foundation for further exploring the molecular mechanism of DNA methyltransferase in regulating the pathogenicity of P. aeruginosa.

Methods
Table S3 In vivo and In vitro enzyme activity analysis using LC-MS/MS.

dA, 2’-deoxyadenosine; 6mdA, N6-methyl-2’-deoxyadenosine, 6mdA/dA, the degree of modifying activity was estimated by the relative abundance of 6mdA normalized by the dA, the abundance of each type of base was calculated from the peak area. The LC-MS/MS measurements were performed on a Shimadzu Nexera high-performance liquid chromatography (HPLC) system (SHIMADZU) coupled with a QTrap5500 triple quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source (Thermo Scientific Q Exactive). Mass spectrums were analyzed by software Xcalibur. The abundance of each type of base was calculated from the peak area, the ratio was calculated by Microsoft Excel.

Table S4. motifs.csv files generated by SMRTLink analysis for strain TBCF and TBCFΔcp respectively.

Table S5. List of significantly differentially regulated genes (DEGs) in TBCFΔcp compared to TBCF, with the location of CP methylation sites and nearby TFBS.

Table S6. Results of GO and KEGG pathway enrichment analysis for DEGs in TBCFΔcp compared to TBCF generated by DAVID.

Table S7. The Closest Neighbors of MTase CP found by REBase. Top 30 BLASTP result is shown.

Table S8. Methylated sites and nearby RpoN binding sites in DEGs.

Table S9. Methylated sites and nearby Dnr binding sites in DEGs.

Appendix A. DNA sequences containing recognition motifs of MTase CP used for in vitro enzyme analysis. Recognition motifs are underlined and methylated adenines are highlighted in grey.