Systematic dissection of σ⁷⁰ sequence diversity and function in bacteria.

Area of Science:

• Bacterial genetics and molecular biology
• Genomics and evolutionary biology

Background:

• Primary sigma 70 (σ⁷⁰) factors are essential conserved bacterial proteins controlling gene expression by binding regulatory DNA.
• The functional implications of sequence diversity among σ⁷⁰ factors across different bacterial species remain largely unexplored.

Purpose of the Study:

• To investigate the relationship between sequence variation and function in primary σ⁷⁰ factors.
• To assess the functional impact of natural σ⁷⁰ sequence diversity within a bacterial context.

Main Methods:

• Utilized multiplex automated genome engineering and deep sequencing (MAGE-seq) to create a saturation mutagenesis library and fitness map for *E. coli* σ⁷⁰ (domains 2-4).
• Employed comparative genomics to map natural σ⁷⁰ sequence diversity onto the *E. coli* fitness landscape.
• Conducted multiplexed transcriptional reporter assays and RNA sequencing (RNA-seq) to evaluate the function of various σ⁷⁰ orthologs.

Main Results:

• Generated a high-resolution fitness map for *E. coli* σ⁷⁰, revealing fitness consequences of mutations across key domains.
• Mapping natural σ⁷⁰ ortholog sequences onto the *E. coli* fitness landscape predicted significant fitness deficits.
• Observed fitness changes for introduced σ⁷⁰ orthologs were generally smaller than predicted deficits, suggesting evolutionary adaptations or compensatory mechanisms.

Conclusions:

• Sequence diversity in bacterial σ⁷⁰ factors is associated with predicted functional differences and potential fitness costs.
• The study provides a framework for understanding σ⁷⁰ evolution and offers insights for engineering these global regulators.