A Noncanonical Intrinsic Terminator in the HicAB Toxin‒Antitoxin Operon Promotes the Transmission of Conjugative Antibiotic Resistance Plasmids

Conjugative plasmids, major vehicles for the spread of antibiotic resistance genes, often contain multiple toxin‒antitoxin (TA) systems. However, the physiological functions of TA systems remain obscure. By studying TA families commonly found on colistin-resistant IncI2 mcr-1-bearing plasmids, we discovered that the HicAB TA, acts as a crucial addiction module to increase horizontal plasmid‒plasmid competition. Overnight cultures of K-12/p45 were diluted to an initial OD600 of approximately 0.1 and then cultivated in LB for 6 h at 37 °C. Total RNA was extracted from the cells via TRIzol® Reagent following the manufacturer’s instructions (Invitrogen), with the genomic DNA removed via the addition of 10 μL of 5 U/μL DNase I (Takara). The RNA quality was subsequently determined using a 2100 Bioanalyzer (Agilent) and quantified using an ND-2000 (NanoDrop Technologies). High-quality RNAs (RIN>8) were utilized for the construction of sequencing libraries. The RNA-seq strand-specific libraries were prepared using a TruSeq RNA Sample Preparation Kit from Illumina (San Diego, CA) with 5 μg of total RNA. The process involved removing rRNA with the RiboZero rRNA removal kit (Epicenter), followed by fragmentation using fragmentation buffer. Further steps included cDNA synthesis, end repair, A-base addition, and ligation of Illumina-indexed adaptors, which strictly followed Illumina's protocol. Libraries were then size selected for cDNA target fragments of 200–300 bp on 2% low-range ultra agarose followed by PCR amplification via Phusion DNA polymerase (NEB) for 15 PCR cycles. After quantification with TBS380, paired-end libraries were subjected to sequencing using the Illumina NovaSeq 6000 platform (Shanghai BIOZERON Co., Ltd.).