Mutations identified in engineered Escherichia coli with a reduced genome

Area of Science:

• Microbiology
• Genomics
• Synthetic Biology

Background:

• Understanding cell growth and survival genes in model organisms like E. coli is key to deciphering complex biological systems.
• Genome reduction strategies, by deleting nonessential genes, offer a powerful approach to study essential genetic functions and cellular adaptations.
• Adaptive laboratory evolution (ALE) is a valuable tool for isolating strains with improved fitness and identifying compensatory mutations.

Purpose of the Study:

• To construct and characterize genome-reduced E. coli strains with significant chromosomal deletions.
• To investigate genetic alterations, including Single Nucleotide Variants (SNVs), insertions, deletions, and inversions, that arise during genome reduction and ALE.
• To identify novel genes involved in cell survival and stress response through the analysis of ALE-rescued strains.

Main Methods:

• Construction of E. coli strains with large deletions spanning approximately 38.9% of the chromosome.
• Employing adaptive laboratory evolution (ALE) to restore partially impaired growth in selected genome-reduced strains.
• Comprehensive genome sequencing of wild-type, genome-reduced, and ALE-evolved strains to identify genetic variations.
• Functional characterization of candidate genes, such as hcaT, using defined deletion mutants.

Main Results:

• Successfully generated genome-reduced E. coli strains with extensive deletions.
• Identified various genetic alterations including SNVs, insertions, deletions, and inversions in both constructed and ALE strains.
• Discovered specific mutations in ALE strains, such as promoter insertions in pntA and IS element in sibE, affecting gene expression.
• Identified a conserved SNV in the hcaT promoter across multiple ALE strains, leading to increased hcaT expression and suggesting a role in oxidative stress survival.

Conclusions:

• Genome reduction and ALE are effective strategies for uncovering essential genes and adaptive mechanisms.
• The study identified mutations that accumulate during genome reduction and ALE, providing insights into genome stability.
• hcaT, a 3-phenylpropionate transporter, was identified as a novel gene involved in stationary phase survival under oxidative stress.
• This research highlights the utility of combining genome engineering with ALE for discovering genes critical for microbial resilience and survival.