Global transcription factors analyses reveal hierarchy and synergism of regulatory networks and master virulence regulators in Pseudomonas aeruginosa

Reviewer #1 (Public review):

Summary:
This work done by Huang et.al. revealed the complex regulatory functions and transcription network of 172 unknown transcription factors of Pseudomonas aeruginosa PAO1. The authors utilized ChIP-seq to profile TFs binding site information across the genome, demonstrating diverse regulatory relationships among them via hierarchical networks with three levels. They further constructed thirteen ternary regulatory motifs in small subs and co-association atlas with 7 core associated clusters. The study also uncovered 24 virulence-related master regulators. The pan-genome analysis uncovered both the conservation and evolution of TFs with P. aeruginosa complex and related species. Furthermore, they established a web-based database combining both existing and novel data from HT-SELEX and ChIP-seq to provide TF binding site information. This study offered valuable insights into studying transcription regulatory networks in P. aeruginosa and other microbes.

Strengths:
The results are presented with clarity, supported by well-organized figures and tables that not only illustrate the study's findings but also enhance the understanding of complex data patterns.

Weaknesses:
The results of this manuscript are mainly presented in systematic figures and tables. Some of the results need to be discussed as an illustration how readers can utilize these datasets.

Reviewer #2 (Public review):

In this work, the authors comprehensively describe the transcriptional regulatory network of Pseudomonas aeruginosa through the analysis of transcription factor binding characteristics. They reveal the hierarchical structure of the network through ChIP-seq, categorizing transcription factors into top-, middle-, and bottom-level, and reveal a diverse set of relationships among the transcription factors. Additionally, the authors conduct a pangenome analysis across the Pseudomonas aeruginosa species complex as well as other species to study the evolution of transcription factors. Moreover, the authors present a database with new and existing data to enable the storage and search of transcription factor binding sites. The findings of this study broaden our knowledge on the transcriptome of P. aeruginosa.

This study sheds light on the complex interconnections between various cellular functions that contribute to the pathogenicity of P. aeruginosa, along with the associated regulatory mechanisms. Certain findings, such as the regulatory tendencies of DNA-binding domain-types, provides valuable insights on the possible functions of uncharacterized transcription factors and new functions of those that have already been characterized. The techniques used hold great potential for discovery of transcription factor functions in understudied organisms as well.

The study would benefit from a more clear discussion on the implications of various findings, such as binding preferences, regulatory preferences, and the link between regulatory crosstalk and virulence. Additionally, the pangenome analysis would be furthered through a discussion of the divergence of the transcription factors of P. aeruginosa PAO1across species in relation to the findings on the hierarchical structure of the transcriptional regulatory network.