Iron content measurement using ICP-MS

Overnight cultures of V. cholerae strains were 1:100 sub-cultured into 250 ml fresh M9 medium, containing 0.2% casein acid hydrolysate as sole carbon source, and grown at 37°C and 200 rpm until OD₆₀₀ reached around 0.4. Then, 0.5 mM IPTG and 0.02% arabinose were added to induce the expression of V. cholerae cbs and dps, respectively. After 4 hrs’ incubation at 30°C and 200 rpm, an aliquot (1 ml) from each culture were taken for enumeration of bacterial cells, while the rest of cells were collected by high-speed centrifugation and stored immediately at -80°C. Three biological replicates were set for each strain.

Bacterial cell pellets were frozen, freeze-dried, and homogenized. Samples (~0.1 g) were weighed into polytetrafluoroethylene vessels, with 6 ml of HNO₃ and 2 ml of 30% H₂O₂, for microwave digestion at 185°C, using the ETHOS E microwave digestion system (Milestone, Italy). The digestion solutions were filtered through quantitative filter papers and quantitatively adjusted to 10 ml with ultrapure water. The iron content in samples was measured by ICP-MS analysis of ⁵⁷Fe (Perkin-Elmer, ELAN DRC-e, USA), and normalized with cell numbers.