RNA isolation and RT-qPCR.

To analyze the relative expression levels of genes within the GAG cluster under normal growth conditions, WT and Tet-somA strains were incubated in MM for 24 h at 37°C. To analyze the relative gene expression levels of the GAG cluster and chitin synthase genes under conditions of CFW stress, WT and Tet-somA strains were incubated in MM for 22 h, and then the samples were supplemented with 100 μg/ml CFW for 0.5, 1, or 2 h. To induce the expression of somA in the Tet-somA mutant, the medium was supplemented with 1 μg/ml doxycycline. The samples were collected and subsequently frozen using liquid nitrogen. Total RNA was isolated using UNIQ-10 column total RNA purification kit (Shanghai Sangon Biotech) according to the manufacturer’s instructions. For gDNA digestion and cDNA synthesis, the HiScriptII Q RT SuperMix for qRCR (+gDNA wiper) kit (Vazyme) were used according to the manufacturer’s instructions. To analyze the relative expression of the interest genes, the resulting cDNAs were used for quantitative PCR, performed with an ABI one-step fast thermocycler (Applied Biosystems) and AceQ qPCR SYBR green master mix (Vazyme). The results were then normalized to tubA, and expression levels were calculated using the ΔΔC_T method (46).