Strains 110spc4 (wild type), 3563 (ΔcoxB::aphII) (19), 6611 (ΔpcuABCDE::aphII) (20), 6611-33 (ΔpcuABCDE::aphII + pcuABCDE), 6611-34 (ΔpcuABCDE::aphII + pcuABDE) [both (20)], 6611-74 (ΔpcuABCDE::aphII + pcuAB[C3M]DE), 6611-75 (ΔpcuABCDE::aphII + pcuAB[CΔC-term]′DE), 6611-76 (ΔpcuABCDE::aphII + pcuABC), and 6611-1696 (ΔpcuABCDE::aphII + pcuAB[C3M, ΔC-term]′DE) were constructed according to standard protocols (20) and grown at 30°C in V3S minimal medium (9.9 mM K2HPO4, 10.1 mM NaH2PO4, 10.0 mM NH4NO3, 3.3 mM MgSO4, 340 μM CaCl2, 59 μM MnSO4, 49 μM H3BO4, 7 μM ZnSO4, 1 μM NaMoO4, 5 μM KI, 5 μM NaSeO3, 9 μM FeCl3, 9 μM FeCl2, 160 nM CuSO4, 105 nM CoCl2, and 105 nM NiCl2) supplemented with 25 mM Na2-succinate and appropriate antibiotics to mid-exponential growth phase, harvested, washed twice with sterile 0.9% NaCl, and transferred into fresh V3S without CuSO4 to a starting OD600 of 0.05. Strains were grown in the presence of spectinomycin (100 μg/ml) for 48 hours at 30°C to OD600 of 0.8. Bacteria were harvested, washed with copper-free V3S, centrifuged, and adjusted to OD600 of 7.0. Identical amounts (15 μl) of each suspension were immediately spotted onto a filter paper soaked in 1% TMPD, and indophenol blue formation was recorded after 10 to 15 min.

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