Hitchhiking genes and boundaries of AmpC amplicons during amoxicillin resistance development in E. coli

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.