Ethics Statement The present research does not involve human subjects, human material, human data, or animals. Methods Strains and growth conditions Bacterial strains are shown in Table 2. Mutations and copA-lacZ transcriptional fusion were transduced by P1 vir lysates into MC4100 strain. Cells were grown aerobically at 37°C with linear shaking in the saline minimal media, MT (2 mM phosphate) [36] and MT + P (defined as MT containing 40 mM phosphate buffer pH 7) [23]. Media were supplemented with 0.5% glycerol and 0.1% tryptone. Growth was monitored by measuring OD560 nm. CB-5083 cost When required, the following antibiotics were used: 100 μg mL−1 ampicillin, 30 μg mL−1 chloramphenicol

and 50 μg mL−1 kanamycin. Table 2 E. coli strains and plasmids used in this work Strains and plasmids Relevant genotype or description Construction or reference MC4100 araD, lac, rpsL, flbB, deoC, ptsF, rbsR, relA1 [37] LSB022 MC4100 (ppkppx::Km) [22] LSB022/pBC29 LSB022/pBC29((ppkppx::Km /ppk + , Ap) [29] RKP2935 RKP4353 [Φ(pitA–lacZ)] pitA::Cm [38] AN3901 JC7623 pitB::Cm [39] AN4080 pitA1 pitB::Cm [39] LSB026 MC4100 pitA:: Cm (P1(RKP2935)xMC4100)

MGP001 MC4100 pitB::Cm (P1(AN3901)xMC4100) JW0473-3 F-, araD-araB, lacZ, copA::km λ − , rph-1, rhaD-rhaB, hsdR CGSC MGP002 MC4100 copA::Km (P1(JW0473-3)xMC4100) MGP003 MGP002 copA::FRT This study MGP004 MGP003 ppkppx::Km, copA − (P1(LSB022)xMGP003) MGP005 MGP004 /pBC29 ((ppkppx::Km, copA − /ppk + , Ap) This study pBC29 (ppk + , Ap) [24] pCP20 Ap, Cm, cI857 lPR flp pSC101 oriTS [40] Cu2+ tolerance Repotrectinib cell line determination buy SB525334 Cells grown in MT and MT + P during

6, 24 or 48 h were incubated with shaking at 37°C for 1 h with different CuSO4 concentrations in the same culture media. Identical aliquots of cells incubated without copper were used as controls. Then, metal tolerance was evaluated by qualitative viability assays, spotting 1/10 serial dilutions on LB-agar [21]. Plates were incubated for 24 h at 37°C. PolyP level measurement Intracellular polyP was measured in cell suspensions by a fluorescence approach using 4′,6-diamidino-2-phenylindole (DAPI) [41]. Briefly, cells were washed and resuspended in T buffer (100 mM Tris–HCl, pH 8). 17 μM DAPI G protein-coupled receptor kinase (Sigma) was added to cuvettes containing cell suspensions (OD560 nm =0.02) in T buffer, with 0.075% SDS and chloroform for cell permeabilization [29]. After 5 min at 37°C with agitation, the DAPI fluorescence spectra (excitation, 415 nm; emission, 445–650 nm) were recorded using an ISS PCI spectrofluorometer (ISS Inc., Champaign, IL). Fluorescence of the DAPI-polyP complex at 550 nm was used as a measurement of intracellular polyP, since emissions from free DAPI and DAPI-DNA are minimal at this wavelength [41]. Membrane electrical potential measurement Changes in the transmembrane electrical potential (ΔΨ) were measured utilizing the potential sensitive fluorescent probe 3,3′-dipropylthiadicarbocyanine (DisC3 [5]) [42].