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Wild-Type LB High Mg Wild-Type Pseudomonas Aeruginosa Wild-type Pseudomonas aeruginosa LB A 0.6 E. coli (LB) B 0.8 P. aeruginosa (LB) 0.6 0.4 0 0 590 0 0 590 6 6 IC =3.0 μM 0.4 D D 50 OD OD O O IC50=7.2 μM 0.2 MIC=32 μM MIC=8 μM 0.2 0.0 0.0 0.1 1 10 100 0.1 1 10 100 TAT-RasGAP [M] TAT-RasGAP [M] WildTa-tt-RyapsGeA PB [MM317]2-326 high MgPseudomonaTast-R aaseGrAuP [gMin317] o-326sa BM2 high Mg E. coli (BM2 Mghigh) P. aeruginosa (BM2 Mghigh) C 0.6 D 1.0 0.8 0.4 0 0 0.6 0 590 590 0 IC =2.0 μM 50 6 6 D D OD OD O IC =10.1 μM O 0.4 50 0.2 MIC=4 μM MIC=32 μM 0.2 0.0 0.0 0.1 1 10 100 0.1 1 10 100 TAT-RasGAP [M] TAT-RasGAP [M] WildTa-t-tRyaspGeAP B [M317] 2-326 low MgPseudomonaTsa t-aRaesrGuAPg [inM317]o-326sa BM2 low Mg v2 E E. coli (BM2 Mglow) F P. aeruginosa (BM2 Mglow) 0.5 0.4 0.4 0.3 0 0.3 0 0 0 590 590 6 6 0.2 IC =1.7 μM D IC =0.3 μM D 50 OD OD O 0.2 50 O MIC=4 μM MIC=0.5 μM 0.1 0.1 0.0 0.0 0.1 1 10 100 0.1 1 10 100 TAT-RasGAP [M] TAT-RasGAP [M] Tat-RasGAP [M317] -326 Tat-RasGAP [M317] -326 Wild-type LB high Mg Pseudomonas aeruginosa LB high Mg G E. coli (LB Mghigh) H P. aeruginosa (LB Mghigh) 0.6 1.5 0.4 1.0 0 0 0 590 590 0 6 6 D IC =16.3 μM D IC =20.8 μM OD OD O 50 50 0.2 O MIC=32 μM 0.5 MIC=64 μM 0.0 0.0 0.1 1 10 100 0.1 1 10 100 Tat-RasGAP [M] TaTATt-Ra-sRasGAPGAP [M] [M] TAT-RasGAP317-326 [M] 317-326 Figure S1. E. coli MG1655 and P. aeruginosa PA14 sensitivity to TAT- RasGAP317-326 varies depending on the growth medium used. E. coli MG1655 or P. aeruginosa PA14 were grown overnight in the indicated medium, diluted to OD600 = 0.1 and grown for 1 hour. Culture was then diluted 20 times and 10 µl was added per well of a 96-well plate containing serial dilutions of TAT-RasGAP317-326. OD590 measurements after 16 hours of incubation in presence of the indicated concentrations of TAT-RasGAP317-326 are shown and MIC is defined as the lowest concentration of TAT-RasGAP317-326 that completely inhibits bacterial proliferation. IC50 is defined as the concentration required to inhibit 50% of growth and was calculated using GraphPad Prism 8. Indicated strains were grown overnight and high diluted respectively in LB (A-B), BM2 with 2 mM MgSO4 (BM2 Mg ) (C-D), BM2 low high with 20 µM MgSO4 (BM2 Mg ) (E-F) or LB with 2 mM MgSO4 (LB Mg ) (G-H). Pseudomonas aeruginosa BMP2s e Hudigohm Mogn aPso alyemruygxiinnosa BM2 low Mg Polymyxin A B 1.0 P. aeruginosa (BM2 Mghigh) 0.3 P. aeruginosa (BM2 Mglow) 0.8 0.2 0 0 0.6 590 0 590 0 6 6 IC =0.45 μg/ml D D 50 IC =1.0 μg/ml OD OD 50 O O 0.4 MIC=1 μg/ml 0.1 MIC=4 μg/ml 0.2 0.0 0.0 0.1 1 10 100 0.1 1 10 100 Wild-type PBolMyPolymyxinm2yx ihn iBg [uhgB / m[Mlg/ml]]g PolymWyixldin-t yBpeP BolyMPolymyxinmy2xi nl oB w[ugB /M m[lg/ml]]g Polymyxin B C D 0.3 E. coli (BM2 Mghigh) 0.3 E. coli (BM2 Mglow) 0.2 0.2 0 0 590 0 590 0 6 6 D D OD OD IC =0.8 μg/ml 50 O O 0.1 0.1 MIC=2 μg/ml IC50=1.0 μg/ml MIC=2 μg/ml 0.0 0.0 0.1 1 10 100 0.1 1 10 100 Tat-RPolymyxinasGAP [MB] [g/ml] Tat-RPolymyxinasGAP [MB] [g/ml] Figure S2. P. aeruginosa but not E. coli grown in medium with high Mg2+ concentration has increased susceptibility to polymyxin B. (A-D) OD590 measurements after 16 hours of incubation in presence of the indicated concentrations of TAT-RasGAP317-326 are shown and MIC is defined as the lowest concentration of TAT-RasGAP317-326 that completely inhibits bacterial proliferation. IC50 is defined as the concentration required to inhibit 50% of growth and was calculated using GraphPad Prism 8. E. coli MG1655 or P. aeruginosa PA14 were grown in BM2 with either 2 mM (High) or 20 M (Low) MgSO4. P. aeruginosa MES pH 5.5 No drug A 4 5 uM TAT-RasGAP MES pH 5.5 E. coli LB untreatedNo drug 10 uM TAT-RasGAP 3 5 55 uMM TAT-RasGAP 20 uM TAT-RasGAP317-326 0 1010 uMM TAT-RasGAP 0 4 600 6 2 D 2020 uMM TAT-RasGAP 0 OD 317-326 O 0 3 6 D O 2 1 1 0 0 0 2 4 6 0 2 4 6 Hours Hours Time [h] P. aeruginosa MES pH 6 P. aeruginosNa oM dErSu gpH 6.5 No drug B C 4 MES pH 6.0 4 MES pH5 u 6.5M TAT-RasGAP 5 uM TAT-RasGAP 10 uM TAT-RasGAP 10 uM TAT-RasGAP 3 3 20 uM TAT-RasGAP317-326 20 uM TAT-RasGAP317-326 0 0 0 0 600 600 6 6 2 2 D D OD OD O O 1 1 0 0 0 2 4 6 0 2 4 6 Hours Hours Time [h] Time [h] P. aeruginosa MES pH 7 P. aeruginoNso ad rLugB pH 7 No drug D E 4 MES pH 7.0 4 Non-buffered5 uM pHTA 7.0T-RasGAP 5 uM TAT-RasGAP 10 uM TAT-RasGAP 10 uM TAT-RasGAP 3 3 20 uM TAT-RasGAP317-326 20 uM TAT-RasGAP317-326 0 0 0 600 0 600 6 2 6 2 D D OD OD O O 1 1 0 0 0 2 4 6 0 2 4 6 Hours Hours Time [h] Time [h] Figure S3. Impact of pH on TAT-RasGAP317-326 activity against P. aeruginosa. (A-E) Growth curves of P. aeruginosa PA14 in LB medium with different pH levels and buffered with 100 mM MES. Bacterial cultures were exposed to different concentrations of TAT-RasGAP317-326 and OD600 was measured to monitor bacterial growth. E. coli LB No drug 5 5 uM TAT-RasGAP 10 uM TAT-RasGAP 4 Preincubation at pH 5.5 No drug 20 uM TAT-RasGAP 0 317-326 0 3 6 A 2.5 B 5 uM TAT (preincub pH 5.5) D Preincubation at pH 5.5 O 2 untreated 10 uM TAT (preincub pH 5.5) 1 2.0 5 M Brightfield Fluorescence 0 20 uM TAT (preincub pH 5.5) 1.5 10 M 0 0 0 600 2 4 6 6 20 M D OD O Hours 1.0 0.5 pH 7.0 0.0 0 2 4 6 Hours PreincuTbiamtei o[hn] at pH 7 No drug 74.1% 2.5 Preincubation at pH 7.0 5 uM TAT (preincub pH 7) 2.0 10 uM TAT (preincub pH 7) 20 uM TAT (preincub pH 7) 1.5 0 0 600 6 pH 5.5 D O OD 1.0 0.5 6.5% 0.0 0 2 4 6 Hours Time [h] Figure S4. Effect of pH on TAT-RasGAP317-326 peptide. (A) TAT-RasGAP317-326 was preincubated in LB pH 5.5 or pH 7.0 for 1 hour. Then, preincubated peptide was added at the indicated concentrations to cultures of E. coli MG1655 and OD600 was measured to monitor bacterial growth. (B) E. coli MG1655 cultures in LB medium at pH 7.0 or 5.5 were incubated with 20 M FITC-TAT-RasGAP317-326 for 6 hours. Cells were then observed by microscopy and percentage of fluorescent cells was quantified. Bar = 5 m. P. aeruginosa MES pH 5.5 E. coNlio M dErSug pH 5.5 No drug E. coli LB A No drug D 4 0.25 ug/ml Polymyxin B 3 0.E.2 5coli ug/ml Polymyxin B 5 P. aeruginosa 5 uM TAT-RasGAP MES pH 5.5 MES pH 5.5 10 uM TAT-RasGAP 0.5 ug/ml Polymyxin B 0.5 ug/ml Polymyxin B 4 untreated 3 20 uM TAT-RasGAP 0 2 317-326 1 ug/ml Polymyxin B 1 ug/ml Polymyxin B 0 3 0.25 g/ml 6 0 0 0 D 0 600 600 6 6 2 O 0.5 g/ml 2 D D O OD OD O 1 g/ml 1 1 1 0 0 2 4 6 Hour0s 0 0 2 4 6 0 2 4 6 Hours Hours P. aerugiTniomsea [h M] ES pH 7 E. coNliT oMi mdeEr u[Shg] pH 7 No drug B E 3 P. aeruginosa 4 0E..2 coli5 ug/ml Polymyxin B 0.25 ug/ml Polymyxin B MES pH 7.0 MES0.5 pH u g7.0/ml Polymyxin B 0.5 ug/ml Polymyxin B 3 2 1 ug/ml Polymyxin B 1 ug/ml Polymyxin B 0 0 0 600 0 6 6 600 2 D D OD O O OD 1 1 0 0 0 2 4 6 0 2 4 6 Hours Hours Time [h] Time [h] P.
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    Supplementary information (a) (b) Figure S1. Resistant (a) and sensitive (b) gene scores plotted against subsystems involved in cell regulation. The small circles represent the individual hits and the large circles represent the mean of each subsystem. Each individual score signifies the mean of 12 trials – three biological and four technical. The p-value was calculated as a two-tailed t-test and significance was determined using the Benjamini-Hochberg procedure; false discovery rate was selected to be 0.1. Plots constructed using Pathway Tools, Omics Dashboard. Figure S2. Connectivity map displaying the predicted functional associations between the silver-resistant gene hits; disconnected gene hits not shown. The thicknesses of the lines indicate the degree of confidence prediction for the given interaction, based on fusion, co-occurrence, experimental and co-expression data. Figure produced using STRING (version 10.5) and a medium confidence score (approximate probability) of 0.4. Figure S3. Connectivity map displaying the predicted functional associations between the silver-sensitive gene hits; disconnected gene hits not shown. The thicknesses of the lines indicate the degree of confidence prediction for the given interaction, based on fusion, co-occurrence, experimental and co-expression data. Figure produced using STRING (version 10.5) and a medium confidence score (approximate probability) of 0.4. Figure S4. Metabolic overview of the pathways in Escherichia coli. The pathways involved in silver-resistance are coloured according to respective normalized score. Each individual score represents the mean of 12 trials – three biological and four technical. Amino acid – upward pointing triangle, carbohydrate – square, proteins – diamond, purines – vertical ellipse, cofactor – downward pointing triangle, tRNA – tee, and other – circle.
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