Re-engineering cellular physiology by rewiring high-level global regulatory genes

Area of Science:

• Microbiology
• Synthetic Biology
• Bacterial Genetics

Background:

• Global regulatory networks control bacterial gene expression and adaptation.
• The hns and stpA genes in Salmonella Typhimurium are key regulators of hundreds of target genes, including those involved in stress resistance.
• Understanding these networks allows for targeted manipulation of bacterial physiology.

Purpose of the Study:

• To investigate the functional consequences of swapping the chromosomal locations and regulatory control of the paralogous hns and stpA genes in Salmonella Typhimurium.
• To engineer a novel bacterial strain with enhanced competitive fitness and tuneable gene expression.
• To explore artificial manipulation of microbial physiology through global regulator rewiring.

Main Methods:

• Reciprocal exchange of the hns and stpA open reading frames and their associated transcription control signals.
• Comparative analysis of the resulting bacterial strain against the wild type, focusing on gene expression patterns and physiological traits.
• Assessment of compensatory mutations and stress response pathways, including the RpoS regulon.

Main Results:

• The engineered strain exhibited superior competitive fitness compared to the wild type, without typical compensatory mutations associated with hns alterations.
• Rescheduled expression of the stress and stationary phase sigma factor RpoS and its regulon was observed.
• Demonstrated successful artificial adjustment of global regulator expression patterns to manipulate microbial physiology.

Conclusions:

• Artificial manipulation of global regulator expression patterns can effectively rewire bacterial gene control programs.
• This approach enables the creation of novel, resilient bacterial organisms with enhanced and tuneable physiological characteristics.
• The study highlights the potential of synthetic biology for engineering microbial fitness and stress resistance.