Systematic discovery of transcription factors and regulatory roles in bacteria
Transcription factors (TFs) control gene expression by direct or indirect activation or repression of target genes. The microorganisms adapt to changing environments through the gene expression. It is necessary to discover and characterize all the TFs in the bacteria. However, the current knowledge on how TFs regulate the target genes in the transcriptional regulatory networks (TRNs) has remained limited due to an incomplete knowledge of the TFs in the bacteria. With the availability of genome-scale experiments and high-throughput sequencing, scientists have made tremendous advances in deciphering the details of the TFs and structures of the TRNs at the system level. For example, Chromatin immunoprecipitation sequencing and Chromatin immunoprecipitation combined with lambda exonuclease digestion (ChIP-seq/ChIP-exo) has enabled researchers to identify DNA binding sites for transcription factors (TFs) on a genome-wide basis. The application of next-generation sequencing (NGS) provides an affordable approach to generate a large number of datasets, which give insight into the understanding of transcriptional regulation networks (TRNs). In this dissertation, I am interested in applying genome-wide approaches (ChIP-exo and RNA-seq) to characterize the binding specificity of TFs and explore the hierarchy structure of TRNs. First, A pipeline to identify the uncharacterized TFs is developed and applied to the model strain Escherichia coli K-12 MG1655. Next, a high-throughput ChIP-exo method are utilized to validate the candidate TFs and discover the binding specificity at the genome in Escherichia coli K-12. In addition, to overcome the challenge of DNA-manipulation in gram-positive strain, monoclonal antibody for transcriptional factor CodY is generated and applied to identify genome-wide binding in Staphylococcus aureus. Last, the reconstruction of strain-specific Fur regulon and pan-regulon is utilized to study the conservation of Fur transcription regulation in the Escherichia coli strains. Our data indicate that the gene expression of target genes is disparate, though the binding pattern is highly conserved within E. coli.