The Adaptations of E. coli SM10λpir (pUCP24T) Under Constant Sub‐MIC Gentamicin Treatment

Background Antibiotics, as a selection stress, could trigger specific responses in bacterial pathogens. This study aimed to investigate adaptive changes of E. coli SM10λpir (pUCP24T) under constant treatment of sub‐MIC Gm (gentamicin). Methods E. coli SM10λpir (pUCP24T) underwent continuous passage culture by serial transfer for 50 days on agar plates containing 30 μg/mL Gm to obtain E. coli SM10λpir (pUCP24T)‐E. Two strains were compared for the horizontal gene transfer ability, stability of plasmid pUCP24T, fitness cost, and expression of conjugation‐related genes. Based on whole genome and RNA sequencing data, functional enrichment analysis (GO and KEGG) was conducted, along with analyses of plasmid sequencing depth, SNPs, and differentially expressed genes (DEGs). Results The conjugation frequency of E. coli SM10λpir (pUCP24T)‐E with recipient PAO1 was higher, and its traI expression was significantly upregulated ( p < 0.05). In the same strain, the growth rate and competition index were lower ( p < 0.05); the sequencing depth of plasmid pUCP24T and the relative expression of the rep gene were much higher ( p < 0.05), but the plasmid showed reduced stability. Functional enrichment analysis suggested a possible enhancement of certain physiological processes and metabolic pathways. A total of 1294 DEGs were detected, with obvious upregulation of hycB , hycD , nikE , cspA , and nanA , and obvious downregulation of gadB , gadC , yeiQ , and yjiH , transcription factors ( appY , gadE ), and sRNAs ( arrS , isrC ). Additionally, the expression of aerobic respiratory pathway genes ( cyoABCDE ) in E. coli SM10λpir (pUCP24T)‐E increased significantly ( p < 0.05). Conclusions The enhanced conjugation frequency during adaptation may be attributed to increased expression of the transfer gene traI and an elevated copy number of plasmid pUCP24T. A heavier fitness cost was imposed on the host during this process. Aerobic respiration and metabolic efficiency were likely potentiated. sRNA isrC was hypothesized to inhibit aerobic respiration by targeting the cytochrome bo oxidase subunit cyoD .