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.

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.

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.

motifString: Detected motif sequence for this site such as “GATC”; centerPos: Position in motif of modification (0-based); modificationType: "modification type, such as ""m6A""; fraction: The percent of time this motif is detected as modified in the genome. (Fraction of instances of this motif with modification QV or identification QV above the QV threshold.); nDetected: Number of instances of this motif that are detected as modified. (Number of instances of this motif with modification QV or identification QV above threshold.); nGenome: Number of occurances of this motif in the reference sequence genome; groupTag: "A name identifying the complete double-strand recognition motif. For paired motifs this is “<motifString1>/<motifString2>”, for example “GAGA/TCTC”. For palindromic or unpaired motifs this is the same as motifString; partnerMotifString: motifString of paired motif (motif with reverse-complementary motifString); meanScore: Mean Modification QV of instances of this motif that are detected as modified; meanIpdRatio: Mean IPD ratio of instances of this motif that are detected as modified; meanCoverage: Mean coverage of instances of this motif that are detected as modified; objectiveScore: Score of this motif in the motif finder algorithm. The algorithm considers higher objective scores to be more confidently identified motifs in the genome based on several factors.

Table S5.

locus-tag: locus tag in TBCF Genome; PAO1 homolog: Homolog gene in PAO1; gene product: gene product; log2FC: log2foldchange, condition TBCF ∆CP vs TBCF. Negative value means downregulated in TBCF ∆CP compared to TBCF; padj: BH adjusted p-values; methylated site: location of the CP recognition methylated sites; fraction: methylation fraction of the site; distance: "distance between methylated site and start coding site, negative value means upstream of the start coding site (putative promoter region), positive value means downstream of the start coding site ( in the gene body). "; Trans-acting factors: Binding sites of RpoN or DNR identified by FIMO in gene promoter or coding region. Only matches with a p-value below 0.0001 were reported.

Table S6.

Category: Database; Term: Enriched terms associated with your genes; Count: Gene matched count; %: Percentage; PValue: Fisher Exact/EASE Score; Genes: A given gene list has found in List Total; List Total: Total genes in the list; Pop Hits: A given gene list has found in Pop Total; Pop Total: Population Total; Fold Enrichment: Enrichment score; Bonferroni: The Bonferroni in DAVID is the Bonferroni Šidák p-value (Šidák 1967) which is a technique slightly less conservative than Bonferroni; Benjamini: Benjamini in DAVID requests adjusted p-values by using the linear step-up method of Benjamini and Hochberg; FDR: "FDR in DAVID requests adaptive linear step-up adjusted p-values for approximate control of the false discovery rate, as discussed in Benjamini and Hochberg (2000). Use the lowest slope method to estimate the number of true NULL hypotheses."; Ref: https://david.ncifcrf.gov/content.jsp?file=functional_annotation.html#funcannochart.

Table S7.

#: serial number; Match to: Target matched MTase in REBASE; Length: Length of Target MTase; Type: RM system; Specificity: recognized sequence; PB: PacBio data available; Meth.: Methylation Type; Fused: sequence fused; Score: Blast Score; E-val: E value; Ident: Identity; Subtype: subtype of RM system; Coverage: Sequence Coverage.

Table S8.

Locus Tag: locus tag inTBCF Genome; Homolog in PAO1(Locus_Tag): Homolog gene in PAO1(Locus_Tag); Homolog in PAO1(gene name): Homolog gene in PAO1(gene name); product: gene product; log2FoldChange: log2foldchange, condition TBCF ∆CP vs TBCF. Negative value means downregulated in TBCF ∆CP compared to TBCF.; padj: BH adjusted p-values; Methsite: location of the CP recognition methylated sites; Fraction: methylation fraction of the site; distance: "distance between methylated site and start coding site, negative value means upstream of the start coding site (putative promoter region), positive value means downstream of the start coding site (in the gene body). "; TF: Binding sites of RpoN identified by FIMO; distance between methylated site and TF binding start: "distance between methylated site and TF start binding site, negative value means upstream of the TF start binding site, positive value means downstream of the TF start binding site. "; score: summing the appropriate entries from each column of the position-dependent scoring matrix that represents the motif.; p-value: the probability of a random sequence of the same length as the motif matching that position of the sequence with as good or better a score.; q-value: the false discovery rate if the occurrence is accepted as significant; matched sequence: TF binding sequence.

Table S9.

Locus Tag: locus tag inTBCF Genome; Homolog in PAO1(Locus_Tag): Homolog gene in PAO1(Locus_Tag); Homolog in PAO1(gene name): Homolog gene in PAO1(gene name); product: gene product; log2FoldChange: log2foldchange, condition TBCF ∆CP vs TBCF. Negative value means downregulated in TBCF ∆CP compared to TBCF.; padj: BH adjusted p-values; Methsite: location of the CP recognition methylated sites; Fraction: methylation fraction of the site; distance: "distance between methylated site and start coding site, negative value means upstream of the start coding site (putative promoter region), positive value means downstream of the start coding site (in the gene body). "; TF: Binding sites of DNR identified by FIMO; distance between methylated site and TF binding start: "distance between methylated site and TF start binding site, negative value means upstream of the TF start binding site, positive value means downstream of the TF start binding site. "; score: summing the appropriate entries from each column of the position-dependent scoring matrix that represents the motif.; p-value: the probability of a random sequence of the same length as the motif matching that position of the sequence with as good or better a score.; q-value: the false discovery rate if the occurrence is accepted as significant; matched sequence: TF binding sequence.

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.