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Hitchhiking genes and boundaries of AmpC amplicons during amoxicillin resistance development in E. coli
Luyuan Nong1, Xinyu Wang1, Belinda Koenders1
1Molecular Biology and Microbial Food Safety, Swammerdam Institute of Life Sciences, University of Amsterdam, Amsterdam, The Netherlands.
Background:
Duplication and amplification of the chromosomal beta-lactamase gene ampC is a main mechanism for amoxicillin resistance development in E. coli. When E. coli is exposed to step-wise increasing amoxicillin concentrations, a fragment carrying the ampC gene can be amplified to high levels, resulting in up to 500 fold increased transcription of ampC. The size of the amplicon varies between replicates and it is not clear what determines the size and boundaries of the fragment. This study investigates the hypothesis that there are conserved amplicon boundaries and hitchhiking genes in amplified fragment.
Results:
Seven mutants were constructed with a deletion of conserved flank-site located genes or hitchhiking genes (∆yjeI, ∆sugE, ∆blc, ∆epmA, ∆yjeM, ∆PecnB and ∆ecnB). The amoxicillin adaptation rates of ∆sugE and ∆epmA were significantly lower than wild type during a 22-day evolution. The duplication or amplification of ampC can be observed in all evolved strains. More genomic point mutations were acquired in evolved ∆epmA and ∆yjeM than in the wild type and other evolved mutants.
Conclusions:
The amplification of ampC does not exhibit a fixed hotspot for its boundaries. IS1A frequently serves as an endpoint in ampC duplications in many evolved E. coli strains. The deletion of sugE and epmA genes, which are located near ampC on the chromosome, affects amoxicillin resistance development in E. coli. Hence the hypothesis is mostly falsified.
Insights
Amoxicillin resistance in E. coli involves ampC gene amplification, but amplicon boundaries are not fixed. Deleting nearby sugE and epmA genes impacts resistance development, falsifying the conserved boundary hypothesis.
Area of Science:
- Microbiology
- Genetics
- Molecular Biology
Background:
- Amoxicillin resistance in E. coli is often driven by amplification of the beta-lactamase gene, ampC.
- This gene amplification can lead to a 500-fold increase in ampC transcription.
- The precise boundaries and determining factors of these amplified DNA fragments remain unclear.
Purpose of the Study:
- Investigate the hypothesis of conserved amplicon boundaries and hitchhiking genes associated with ampC amplification.
- Determine if specific genes near ampC influence the rate of amoxicillin resistance development.
Main Methods:
- Constructed seven E. coli mutants with deletions of specific genes flanking or within the ampC amplicon.
- Performed 22-day evolution experiments with stepwise increasing amoxicillin concentrations.
- Analyzed genomic point mutations and gene duplications/amplifications in evolved strains.
Main Results:
- The amplification of ampC did not show fixed boundaries; IS1A was frequently observed as an endpoint.
- Deletion mutants ∆sugE and ∆epmA exhibited significantly reduced amoxicillin adaptation rates compared to wild-type.
- Evolved ∆epmA and ∆yjeM strains acquired more genomic point mutations than wild-type.
Conclusions:
- The hypothesis of conserved amplicon boundaries for ampC amplification is largely falsified.
- IS1A acts as a common endpoint for ampC duplications.
- The genes sugE and epmA, located near ampC, play a role in the development of amoxicillin resistance in E. coli.
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