Korotetskiy, Ilya S.Shilov, Sergey V.Kuznetsova, Tatyana V.Zubenko, NatalyaIvanova, LyudmilaReva, Oleg N.2025-10-242025-10-242025-05Korotetskiy IS, Shilov SV, Kuznetsova TV, Zubenko N, Ivanova L, Reva ON (2025) Epigenetic background of lineage-specific gene expression landscapes of four Staphylococcus aureus hospital isolates. PLoS One 20(5): e0322006. https://doi.org/10.1371/journal.pone.0322006.1932-6203 (online)10.1371/journal.pone.0322006http://hdl.handle.net/2263/104986DATA AVAILABILITY : The genome assemblies, original long DNA reads generated by SMRT PacBio technology, and raw RNA reads are available from the respective BioSample resources at the NCBI database: S. aureus SCAID OTT1-2022 (150) – SAMN30333622 (CP102945); S. aureus SCAID OTT1-2021 (597/2) – SAMN20982456 (chromosome CP082813, plasmid CP082814); S. aureus SCAID WND1-2021 (598) – SAMN20982455 (CP082815); and S. aureus ATCC BAA-39 – SAMN09635552 (CP033505). Software tools designed for this project, together with intermediate input files, including gene expression RPKM values, GFF files with predicted nucleotide modifications identified by the ipdSummary program, and a COG Table with predicted clusters of orthologous groups, are available from the Zenodo public databasehttps://zenodo.org/records/10571089, doi: 105281/zenodo10571089; https://zenodo.org/records/10571197, doi: 105281/zenodo10571197). GRAPHICAL ABSTRACT. This research aims to identify potential links, albeit indirect, between strain-specific gene expression and regulation patterns (A) and patterns of epigenetic modifications of genomic nucleotides (B). (A) Gene expression regulation in S. aureus 150 under the combined effect of two antimicrobials: gentamicin and the iodine complex CC-196. Genes with varying expression levels are plotted as dots based on their baseMean (X-axis) and Log2FoldChange (Y-axis) values. (B) Chromosomal adenines (represented by red dots) in the same strain are plotted according to their sequence depth (coverage) and nucleotide modification (NucMod) calling scores. Adenine residues with NucMod scores above 20 were selected, including those within identified canonical motifs (canonical methylation) and sporadically distributed epigenetically modified adenines (non-canonical modifications). https://doi.org/10.1371/journal.pone.0322006.s001. FIGURE S1. Flowchart diagram of the analytic pipeline designed for elucidation of statistically significant associations between epigenetic modifications of bases in TSC-upstream regions and the level of gene expression. https://doi.org/10.1371/journal.pone.0322006.s002. FIGURES S2. Results of genome alignment using the progressiveMauve algorithm (Mauve v20150226) (A) Alignment of the chromosome sequences of four Staphylococcus aureus strains. Each chromosome is represented as a series of coloured blocks, denoting homologous regions shared among the genomes. These blocks function as histograms, reflecting sequence similarity across the aligned segments Gaps between blocks indicate strain-specific insertions. Numbers above each block represent their respective positions on the chromosomes. (B) Dendrogram depicting relationships based on genomic sequence similarity. https://doi.org/10.1371/journal.pone.0322006.s003. FIGURE S3. Methylation patterns of the genomes of four Staphylococcus aureus strains. (A) S aureus 150, methylation at canonical motifs ACaYNNNNNGGt; (B) S aureus 150: methylation at canonical motifs CGaNNNNNNCtC; (C) S aureus 597/2: methylation at canonical motifs GWaGNNNNNNtAAA; (D) S aureus 598: methylation at canonical motifs GGaNNNNNNNtCG; (E) S aureus 598: methylation at canonical motifs GWaGNNNNNGAt; (F) S aureus BAA-39: methylation at canonical motifs GaYNNNNNNRtGG; (G) S aureus BAA-39: methylation at canonical motifs GTaNNNNNCtTC. Blue triangles indicate the locations of methylated sites. Canonical motif sequences are displayed along the central paths of the genomic atlas views The numbers of methylated versus total canonical motifs found per genome are also shown GC content and GC skew values, calculated over a 5,000 bp sliding window, are depicted by color-coded circular histograms, as explained in the figure legend. https://doi.org/10.1371/journal.pone.0322006.s004. FIGURE S4. Visualization of canonical methylation and non-canonical modifications of adenine residues in the genome of Staphylococcus aureus 597/2. (A) Dot-plot presentation of the distribution of modified adenine residues with NucMod scores above 20 units. Each node represents an individual epigenetically modified adenine residue, plotted according to the estimated coverage at this location and the NucMod score. (B) Distribution of modified adenine residues associated with the canonical DNA methylation motif GWaGNNNNNNtAAA. (C) Distribution of non-canonically modified adenine residues. Panels in the top-left corners of the plots show the total numbers of identified epigenetically modified adenines and the numbers of residues methylated at the 6th carbon (m6A-methylation), as predicted by the program ipdSummary, versus the number of adenine residues with unknown types of modification (modA). https://doi.org/10.1371/journal.pone.0322006.s005. TABLE S1. Homologous genes identified in four selected S aureus strains. https://doi.org/10.1371/journal.pone.0322006.s006. TABLE S2. Transcripts Per Million (TPM) values of the expression of homologous genes in tested Staphylococcus aureus cultures under the following growth conditions: NC – negative control; GE – treated with gentamicin; CC – treated with iodine-containing complex CC-196; GECC – combinatorial treatment. https://doi.org/10.1371/journal.pone.0322006.s007. TABLE S3. Transcripts Per Million (TPM) values of expression of antibiotic resistance and virulence genes found in tested Staphylococcus aureus cultures under the following growth conditions: NC – negative control; GE – treated with gentamicin; CC – treated with iodine-containing complex CC-196; GECC – combinatorial treatment NF – gene not found in the given genome. https://doi.org/10.1371/journal.pone.0322006.s008. TABLE S4. Homologous genes showing strain-specific differential expression in four selected Staphylococcus aureus strains and consistent expression under stress conditions. https://doi.org/10.1371/journal.pone.0322006.s009. TABLE S5. Locations of the RM systems in the chromosomes of the four Staphylococcus aureus strains and the RPKM values of expression of the involved genes. https://doi.org/10.1371/journal.pone.0322006.s010. TABLE S6. Pairs of homologous genes exhibiting alternative methylation in the region from –117 to –81 bp. upstream of the transcription start codons (TSC), belonging to different gene expression categories. Locus tag identifiers of different test cultures are: NW338 – Staphylococcus aureus 150; K8B68 – S. aureus 597/2; K8B78 – S. aureus 598; and HMPRNC0000 – S. aureus BAA-39. https://doi.org/10.1371/journal.pone.0322006.s011.Bacteria with similar genomes can exhibit different phenotypes due to alternative gene expression patterns. In this study, we analysed four antibiotic-resistant Staphylococcus aureus hospital isolates using transcriptomics, PacBio genome sequencing, and methylomics analyses. Transcriptomic data were obtained from cultures exposed to gentamicin, the iodine-alanine complex CC-196, and their combination. We observed strain-specific expression patterns of core and accessory genes that remained stable under antimicrobial stress – a phenomenon we term the Clonal Gene Expression Stability (CGES) that is the main discovery of the paper. An involvement of epigenetic mechanisms in stabilization of the CGES was hypothesized and statistically verified. Canonical methylation patterns controlled by type I restriction-modification systems accounted for ~ 10% of epigenetically modified adenine residues, whereas multiple non-canonically modified adenines were distributed sporadically due to imperfect DNA targeting by methyltransferases. Protein-coding sequences were characterized by a significantly lower frequency of modified nucleotides. Epigenetic modifications near transcription start codons showed a statistically significant negative association with gene expression levels. While the role of epigenetic modifications in gene regulation remains debatable, variations in non-canonical modification patterns may serve as markers of CGES.en© 2025 Korotetskiy et al. This is an open access article distributed under the terms of the Creative Commons Attribution License.Clonal gene expression stability (CGES)Gene expression landscapesStaphylococcus aureusAntibiotic-resistantHospital isolateTranscriptomicsPacBio genome sequencingMethylomics analysesArticle