Fig. 1: Mapping Ag⁺-proteome in S. aureus.

a Minimum inhibitory concentration (MIC) test of AgNO₃ against S. aureus in BHI culture medium. The measured MIC₅₀ of 20 μg/mL (118 μM) AgNO₃ was applied to treat S. aureus for extraction of proteins (n = 3). The OD₆₀₀ of S. aureus cells without treatment of AgNO₃ was set as 100%. The growth of S. aureus treated with different concentrations of AgNO₃ was normalized to that of the control group. b Growth curves of S. aureus after treatment of different concentrations of AgNO₃ (n = 3). c Time-dependent uptake of Ag⁺ in S. aureus cells after treatment with 20 μg/mL AgNO₃ for 5, 30, 60, and 120 min (n = 3). d Distribution of Ag⁺ in different fractions of S. aureus after treatment with 20 μg/mL AgNO₃ for 5, 30, and 60 min (n = 3). The other stands for the pellet that could not be dissolved by 1% SDS, which is mainly composed of the cell wall. e Exploration of Ag⁺-binding proteins in soluble fraction of S. aureus with LC-GE-ICP-MS (n = 3). 31 Ag⁺-associated proteins were observed, separated, and identified. In the 3D figure, the x-axis, y-axis, and z-axis show the isoelectric points of Ag⁺-binding proteins according to the LC separation, the molecular weights that were referenced to the ¹²⁷I-labeled protein markers, and the intensity (counts) of Ag contained in proteins, respectively. 1 (UPF0356), 2 (AhpD core domain), 3 (RplK), 4 (RplI), 5 (AhpD), 6 (Bcp), 7 (Dps), 8 (UspA), 9 (FtnA), 10 (Tnp), 11 (TrxA), 12 (CodY), 13 (RpoA), 14 (CysS), 15 (TrxB), 16 (Ldh1), 17 (PfkA), 18 (PdhB), 19 (CcpA), 20 (Trps), 21 (Pgl), 22 (PepP), 23 (Int), 24 (Hmp), 25 (TelA), 26 (Tuf), 27 (Eno), 28 (Gnd), 29 (Mqo2), 30 (PflB), and 31 (RpoB). f Silver contents in identified Ag⁺-binding proteins. The silver contents were calculated by integrating the peaks of corresponding Ag⁺-binding proteins. g GE-ICP-MS profiles of Ag⁺-RpoA, Ag⁺-Pgl and Ag⁺-Gnd. h MALDI-TOF mass spectra of RpoA and Pgl without and with pre-incubation with Ag⁺. i Gene Ontology (GO) analysis of biological processes enriched by Ag⁺-binding proteins in S. aureus analyzed via STRING. Eleven biological processes were significantly enriched by GO analysis with FDR < 0.05. j Distribution of Ag⁺ in different functional categories. The silver contents in identified proteins were summed according to the proteins involved in different pathways. The sum of silver contained in all identified Ag⁺-binding proteins was set as 100%. (a, b, c) Mean value of three replicates is shown and error bars indicate ±SEM. (e, g, h) One representative of three replicates is presented.