Flavomycin inhibits conjugative transfer of plasmid-mediated antibiotic resistance genes by perturbing energy support and pilus assembly

RNA was extracted using the RNeasy Mini Kit (Qiagen), following the manufacturer’s protocol. The integrity and concentration of RNA were assessed using a NanoDrop spectrophometer (Thermo Fisher Scientific), and genomic DNA contamination was eliminated using RNase-free DNase I (Qiagen, Germany). Subsequently, RNA samples were processed for sequencing on the Illumina HiSeq System(Caporaso et al., 2012), and data analysis was conducted as previously described(Liao et al., 2013). The RNA-Seq data were used to assemble the transcriptome with Trinity RNA-Seq software(Haas et al., 2013), and RNA-Seq reads were aligned to the E. coli BW25113 reference genome (CP009273.1) using the Subread aligner(Liao et al., 2013).Differentially expressed genes (DEGs) were conducted using the Fragments Per Kilobase of transcript per Million mapped reads (FPKM) approach with a false discovery rate (FDR) adjusted to P <0.05 and fold change valves (FC) ≥2. The DEGs were further analyzed for their involvement in biological pathways and functions using several bioinformatics resources, including the Database for Annotation, Visualization and Integrated Discovery (https://david.ncifcrf.gov/)(Huang Da et al., 2009), Kyoto Encyclopedia of Genes and Genomes (KEGG)(Kanehisa and Goto, 2000), and BioCyc(Caspi et al., 2018) databases