Evolutionary and Functional Diversity of Regulatory Factors and Sequences that Coordinate Gene Expression

Bacteria regulate gene expression through coordinated interactions between cis-regulatory sequences and trans-regulatory factors. Understanding the molecular basis for the functions of these regulatory components is not only essential for deciphering complex biological processes in diverse bacteria but also critical for rational engineering of microbial phenotypes. However, systematically dissecting the sequence-function relationships of cis and trans regulatory components that underly gene expression is still a key challenge. Recent technological advances have provided novel tools and methods for mapping sequence-function relationships in high-throughput. This dissertation focuses on applying novel methods enabled through increased throughput and scalability of DNA synthesis and sequencing to elucidate the sequence-function relationships of cis and trans components that underlie bacterial gene regulation. In Chapter 2, evolutionary and functional diversity of primary σ70, a universally conserved global regulator in bacteria, is studied through comparative genomics, saturation mutagenesis, and transcriptomics. Through the combined efforts of these approaches, we demonstrate that sequence diversity of σ70 factors reflects functional differences which have been shaped by evolutionary constraints from co-evolving regulatory sequence targets during evolution. Chapter 3 discusses systematically mapping transcriptional activities of cis-regulatory sequences from Biosynthetic Gene Clusters (BGCs). Using a Streptomyces as a host, we found key regulatory features that affected gene expression, such as GC content, transcription start sites, and sequence motifs. We further explored regulation of BGC derived regulatory sequences by expressing global regulatory factors and screening for regulator sequences with altered expression levels. Finally, Chapter 4 highlights recent studies that made key contributions towards elucidating and modulating bacterial gene regulatory networks and reviews the current state of microbial systems biology and gene regulation. Together, the results and discussions presented in this dissertation seeks to further advance the current knowledge of sequence-function relationships of microbial regulatory components to enable better understanding, modeling, and rational engineering of bacterial gene regulation.