Quantifying Cellular Zn2+ and Ni2+ in K. pneumoniae

The cellular Zn²⁺ and Ni²⁺ content of K. pneumoniae was carried out according to the method described by Maunders et al.^{66 (link)} with some modifications. An overnight culture of K. pneumoniae was diluted 1/200 in LB medium (5 mL) and was either untreated or treated with 0.2 mM AMA or Zn-AMA. The cells were grown for 16 h at 37 °C, harvested by centrifugation, and washed twice with PBS containing 5 mM EDTA and twice with ultra-pure water. The cell pellet was subsequently digested in 0.25 mL 65% HNO₃ at 80 °C for 30 minutes in borosilicate glass tubes using a water bath. The samples were diluted to 2% HNO₃ and filtered through a 0.45 μm syringe filter. ⁶⁰Ni and ⁶⁶Zn were quantified using a Thermo iCAP Q ICP-MS.