Transcriptomic Analysis of K. pneumoniae Zinc Stress

K. pneumoniae strains were grown in Zn(II)-limited or Zn(II)-supplemented media at 37°C with aeration to mid-log phase (OD_{600 =} 0.6-0.8) and harvested by centrifugation at 18,000 × g for 5 min at room temperature. Cells were resuspended in RNA Protect Bacteria Reagent (Qiagen) and total RNA extracted using the RNeasy Mini Kit with two rounds of on-column DNase I digestion, as per manufacturer’s instructions, with final concentrations determined by spectrophotometry. Quantitative real-time-PCR (qRT-PCR) was performed on the QuantStudio 7 Real-Time PCR System using the SuperScript III Platinum SYBR Green One-Step qPCR Mix (ThermoFisher Scientific) using rpoD to normalize gene expression, essentially as described previously (Maunders et al., 2022 (link)). Oligonucleotides used are listed in Supplementary Table 2.