Figure 5. Subcellular localization and quantification of mature fluorescence pyoverdine in the periplasm and cytoplasm.

(a) Localization of mature fluorescence pyoverdine was detected in wild-type, clpV::Tn5, ΔicmF, tssC::Tn5, pvdL::Tn5, and pvdE::Tn5 mutants through a fluorescence microscope with a DAPI filter. (Scale bar = 2 μm). (b) Quantification of periplasmic and cytoplasmic pyoverdine was conducted by measuring fluorescence at excitation 405 nm and emission 460 nm. Pyoverdine values were normalized against the cell optical densities (Ex405, Em 460/OD₆₀₀), and relative values were determined by comparing each value to the periplasmic mean value of the wild-type. Values are mean ± SD of three independent experiments. P. taiwanensis and mutants (clpV::Tn5, ΔicmF, tssC::Tn5, pvdL::Tn5 and pvdE::Tn5) were grown on the iron-limited media for 12 h at 28 °C.