Overview of ChIP-seq results.

(A). Density of all TFs (green) and ChIPed TFs (orange) in this study throughout the P. aeruginosa genome. (B). Annotation heatmap of all peak distribution with 6 locations: Upstream, where the peak is located entirely upstream of the gene; Downstream, where the peak is positioned completely downstream of the gene; Inside, where the peak is entirely contained within the gene body; OverlapStart, where the peak overlaps with the 5’ end of the gene; OverlapEnd, where the peak overlaps with the 3’ end of the gene; and IncludeFeature, where the peak completely encompasses the gene. (C). Peak distance to the translational start site (TSS) of each DBD family. (D). Treemap of the 172 TFs peak numbers based on DBD family. Each box’s size represents the family’s size (number of peaks), and the explained variance of each DBD type means the color shades of each box. DBD families of ChIPed TFs are classified into 20 different categories: LuxR, LysR, Two DBDs (Two DNA binding domains), AraC, TetR, ArsR, CRP, OmpR, GntR, MarR, AsnC, Cro/CI, TyrR, Rrf2, MerR, IclR, Fis, RpiR, DeoR, and undetectable. (E). The dot plot shows the top 10 gene ontology (GO) terms from the PseudoCAP annotation of ChIPed TFs. The size of the dots indicates the significance of each functional category, quantified by −log10 (p. adjust).

Hierarchical networks and network motifs.

(A). Overview of hierarchy regulatory network after removing TFs with degree O+I <= 10. Nodes indicate TFs, and the color represents the hierarchical level. The top level was highlighted in red, the middle in yellow, and the bottom highlighted in blue. The edges with arrows indicate the regulatory direction. Gray means downward-pointing, and red means upward-pointing. (B). The auto-regulator motif of 9 TFs. (C). Three transcription- factor motifs occurrences with five basic triangular motifs and eight toggle switch motifs. The circle presents TF, and the arrow indicates the regulatory direction. (D). Alluvial diagram reveals basic triangle motif 3 (n = 1956) depending on DBD types. The color of splines is highlighted in different DBD families, and the name of DBD families are labeled.

Core co-association regulatory networks.

(A). Core clusters of significant co-binding patterns of TFs. Each TF is highlighted in different colors based on DBD types. The co-association score by pair of TFs was calculated by Jaccard statistics, which measures the ratio of the number of base pairs in overlapped binding peaks on both TFs to the number of base pairs in their union. (B). The histogram’s overlapped target genes of TFs in cluster 3 represent the number of target peaks in the individual/overlapped set. (C). The network of co-regulation of TFs in cluster 3 with co-bound targets of more than 4. (D-E). Genome browser view of TFs in cluster 3 binding intensities at the PA2504 and pqsH locus.

Newly identified virulence-related master regulators.

(A). Overview of all identified master regulators related to 6 pathways, including QS, motility, biofilm, siderophore, pyocyanin, and ROS. Each circle represents one TF, with the height indicating the significance, quantified by -log10 (p.adjust), and the size indicates the number of targets associated with the virulence pathway. (B). Intersection of master regulators in 6 virulence pathways. The bar chart on the top shows the number of intersections, while the matrix below indicates which TFs are involved in specific biological processes such as siderophore production, pyocyanin production, biofilm formation, ROS response, QS, and motility.

Overview of transcriptional network of TFs in P. aeruginosa.

(A). The interaction network of virulence-related master regulators in P. aeruginosa. The ten virulence pathways are highlighted in different colors. The size of the circle represents the degree, and the width of the edges indicates the overlapped number of TFs between two pathways. (B). The target annotated using the COG database shows four orthology classification profiling from different DBD types TFs. The size of rectangle indicates the number of targets.

Conservation and variability of TFs in PAO1.

(A). The pie chart shows the proportions of genes categorized by their presence across P. aeruginosa strains for all genes. (B). The pie chart shows the distribution of TFs identified from PAO1 across different conservation categories. (C). The bar plot of the proportion for non- core TFs. Genes are categorized based on their presence frequency across P. aeruginosa strains: Core genes (present in 99% ∼ 100% strains), Soft core genes (present in 95% ∼ 99% strains), Shell genes (present in 15% ∼ 95% strains), and Cloud genes (present in 0% ∼ 15% strains). (D). The conservation and evolutionary relationship of all 373 TFs in PAO1 among bacteria, archaebacteria, fungi, plants, and animals. The conservation value was normalized after blastp alignment86. The phylogenetic trees were constructed using MEGA1187 and plotted via R package ggtree88.