Transcriptional Analysis of Bacterial Genes

A single colony of each strain was cultured overnight in THYB at 37 °C, and resuspended in the same medium at an OD₆₀₀ of 0.01. Cells were harvested at mid-exponential (OD₆₀₀ = 0.4–0.6) and stationary (OD₆₀₀ = 1.2–1.4) phases and added to two volumes of RNAprotect (Qiagen 76506). Total RNA was then isolated using the RNeasy minikit (Qiagen 74104) and the samples were treated with Turbo DNase (Life Technologies AM1907) to remove any genomic DNA. Conversion of RNA to cDNA was performed using SuperScript VILO cDNA synthesis kit (Invitrogen 11754050). Reverse transcription-quantitative PCR (RT-qPCR) was performed using SYBR green master mix (Applied Biosystems 4309155) according to the manufacturer’s instructions and primers specific for thfT (thfT-qPCR-S1: 5’-GCC AGT GGG TCA GGT AAT TT-3’; thfT-qPCR-A1: 5’-GAC AGT GGT TTC CGG TAG AAG-3’) and gyrA (RTPCR-gyrA-fw: 5’-CGA CTT GTC TGA ACG CCA AA-3’; RTPCR-gyrA-rv: 5’- GTC AGC AAT CAA GGC CAA CA-3’)^{26 (link)}. qPCR was performed on a CFX96 Touch Real-Time PCR machine (BioRad) and analysed using CFX Manager software (BioRad). Relative gene expression was calculated using the threshold cycle (2^−ΔΔCt) method with gyrA as the reference housekeeping gene. All reactions were performed in triplicate from three independently isolated RNA samples for each strain.