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 alignment90. The phylogenetic trees were constructed using MEGA1191 and plotted via R package ggtree92.

RNA-seq for all TFs and ChIPed TFs.

Previous RNA-seq data shows the expression level of all TFs and ChIPed TFs. The circle size indicates the log2-transformation raw counts, and the color highlighted for ChIPed TFs represented different DBD families.

Distribution of Hierarchy Height

h. h = (O-I)/(O+I), O presents the extent they regulate other TFs, and I indicates the level other TFs regulate them. (A). TFs can be divided into almost equal three levels depending on index h: Bottom level (−1.0 < h < 0.75), Middle level (−0.75 < h < 0.75), and Top level (0.75 < h < 1.0). (B). The number of TFs resided in three levels after removing TFs with O+I <10.

Circular phylogenetic tree of TF binding motifs.

(A). Circular phylogenetic tree representing the clustering of TFs based on their motif similarity. The tree was constructed using hierarchical clustering with the Ward.D2 method applied to the pairwise similarity matrix of motifs. The tree were visualized using ggtree93. Each node represents a TF, color-coded by its DBD family classification as indicated in the legend on the right. The motif logos are aligned with the respective TFs, with their orientations adjusted to maintain legibility around the circular layout. (B). PA3587 and MetR display similar DNA binding motifs.

Co-association network of TFs.

(A). Heatmap reveals a full co-association pattern of all TFs. (B). The density of co-association score for all TF pairs. Determining co-association score as 0.1 (dashed in red) of significant TF co-associations based on an elbow statistic.

Validation and co-regulation of virulence-related master regulators.

(A). The validation of the binding sites of PA0167 by EMSA. (B). The validation of the binding sites of PA0815 by EMSA. (C). The validation of the binding sites of PA1380 by EMSA. (D). The validation of the binding sites of PA3094 by EMSA. (E). The detection of expression of target genes PA1380, cupB1, and cupB3 in WT, ΔPA1380 and complementary strain by RT-qPCR. (F). The detection of expression of target genes PA3094 and lecA in WT, ΔPA3094 and complementary strain by RT-qPCR. (G). Graph diagram of interactions involving target genes of 4 TFs, including PA1380, PA0815, PA5428, and PA3973. NC indicates negative control. Student’s t test, *P ≤ 0.05.

Regulators involved in TCA and Ribosome pathways.

(A). Radar plots show the putative regulator in TCA and ribosome pathways. (B). Graph diagram of interactions involving target genes of 14 TFs, including PA0611, PA5218, PA5431, PA1759, PA0403 (PyrR), PA 1283, PA4381, PA5255 (AlgQ), PA4784, PA0475, PA0893, PA0448, PA2957 and PA5438.

Conserved TFs in P. aeruginosa.

(A). The coverage of PhoB and RpoN peak regions over PAO1 and PA14 chromosomes. Each line shows the location and log2 Fold Enrichment of peaks signal in the chromosome. (B). The binding motifs of PhoB and RpoN were analyzed via MEME-ChIP. All peaks were used to define the binding motif. (C). Comparison of overlapped targets enriched in PAO1 and PA14 of PhoB and RpoN. The Fisher test made the significance of the overlap ratio. (D). Phylogenetic tree of PA2032 across different domains of life. The phylogenetic tree was constructed using PA2032 sequence from PAO1 as the root.