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Published on: November 12, 2012
Conditional lethal amber mutations in essential Escherichia coli genes
Christopher D Herring1, Frederick R Blattner
1Laboratory of Genetics, University of Wisconsin-Madison, 445 Henry Mall, Madison, WI 53706, USA.
Abstract:
The essential genes of microorganisms encode biological functions important for survival and thus tend to be of high scientific interest. Drugs that interfere with essential functions are likely to be interesting candidates for antimicrobials. However, these genes are hard to study genetically because knockout mutations in them are by definition inviable. We recently described a conditional mutation system in Escherichia coli that uses a plasmid to produce an amber suppressor tRNA regulated by the arabinose promoter. This suppressor was used here in the construction of amber mutations in seven essential E. coli genes. Amber stop codons were introduced as "tagalong" mutations in the flanking DNA of a downstream antibiotic resistance marker by lambda red recombination. The drug marker was removed by expression of I-SceI meganuclease, leaving a markerless mutation. We demonstrate the method with the genes frr, gcpE, lpxC, map, murA, ppa, and rpsA. We were unable to isolate an amber mutation in ftsZ. Kinetics of cell death and morphological changes were measured following removal of arabinose. As expected given the wide range of cellular mechanisms represented, different mutants showed widely different death curves. All of the mutations were bactericidal except the mutation in gcpE, which was bacteriostatic. The strain carrying an amber mutation in murA was by far the most sensitive, showing rapid killing in nonpermissive medium. The MurA protein is critical for peptidoglycan synthesis and is the target for the antibiotic fosfomycin. Such experiments may inexpensively provide valuable information for the identification and prioritization of targets for antibiotic development.
Insights
Researchers developed a new method to study essential genes in bacteria by creating conditional mutations. This technique helps identify potential new antimicrobial drug targets by observing bacterial responses to gene inactivation.
Area of Science:
- Microbiology
- Genetics
- Molecular Biology
Background:
- Essential genes are crucial for microbial survival and are prime targets for antimicrobial drug development.
- Studying essential genes is challenging due to inviability from knockout mutations.
- A conditional mutation system using an amber suppressor tRNA regulated by the arabinose promoter was previously established.
Purpose of the Study:
- To develop and demonstrate a method for creating markerless conditional mutations in essential Escherichia coli genes.
- To investigate the effects of inactivating essential genes on bacterial viability and morphology.
- To identify potential new antimicrobial targets by assessing the sensitivity of mutants to gene inactivation.
Main Methods:
- Utilized lambda red recombination to introduce amber stop codons into essential E. coli genes, creating 'tagalong' mutations.
- Employed I-SceI meganuclease to remove antibiotic resistance markers, generating markerless mutations.
- Constructed conditional mutations in seven essential genes (frr, gcpE, lpxC, map, murA, ppa, rpsA) and assessed cell death kinetics and morphology upon arabinose removal.
Main Results:
- Successfully created markerless conditional mutations in seven essential E. coli genes.
- Observed varied cell death kinetics and morphological changes across different mutants, indicating diverse cellular roles.
- Identified that mutations in murA resulted in the most rapid killing, highlighting MurA's critical role in peptidoglycan synthesis.
- Found that most mutations were bactericidal, with the exception of gcpE, which was bacteriostatic.
Conclusions:
- The developed method provides an efficient way to study essential genes and their functions in bacteria.
- This approach can accelerate the identification and prioritization of novel antimicrobial drug targets.
- The sensitivity of mutants to gene inactivation offers valuable insights for antibiotic development, as exemplified by the murA mutant and its link to fosfomycin.
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