Systematic dissection of σ(70) sequence diversity and function in bacteria

Primary σ(70) factors are key conserved bacterial regulatory proteins that interact with regulatory DNA to control gene expression. It is, however, poorly understood whether σ(70) sequence diversity in different bacteria reflects functional differences. Here, we employ comparative and functional genomics to explore the sequence and function relationship of primary σ(70). Using multiplex automated genome engineering and deep sequencing (MAGE-seq), we generate a saturation mutagenesis library and high-resolution fitness map of E. coli σ(70) in domains 2–4. Mapping natural σ(70) sequence diversity to the E. coli σ(70) fitness landscape reveals significant predicted fitness deficits across σ(70) orthologs. Interestingly, these predicted deficits are larger than observed fitness changes for 15 σ(70) orthologs introduced into E. coli. Finally, we use a multiplexed transcriptional reporter assay and RNA sequencing (RNA-seq) to explore functional differences of several σ(70) orthologs. This work provides an in-depth analysis of σ(70) sequence and function to improve efforts to understand the evolution and engineering potential of this global regulator.