Methods All growth media, antibiotics and chemicals were purchased from Sigma-Aldrich (Poole, Dorset, UK) unless stated otherwise. Bacterial strains and plasmids E. coli BW25113 [45] and its ΔmdtM and ΔmdfA deletion mutants [46] were obtained from the Keio collection (National BioResource Project, Japan) and used for growth assays. The ΔmdtM and ΔmdfA deletion mutants were used as the background strains for testing alkalitolerance of cells expressing

wild-type mdtM (pMdtM) or dysfunctional MdtM D22A (pD22A) mutant from pBAD/Myc-His A vector (Invitrogen). Construction of these plasmids was described before [24]. The outer membrane permeability mutant E. coli UTL2 [47] was used for whole cell EtBr efflux assays. E. coli TO114 [26], a strain deficient in the Na+/H+ antiporters NhaA and NhaB, and the K+/H+ antiporter ChaA, was complemented

with pMdtM or pD22A and used for production of inverted vesicles for use in transport assays. #Pritelivir order randurls[1|1|,|CHEM1|]# Bacterial growth assays on solid medium Cultures from single bacterial colonies were grown at 37°C to an OD600 of 1.0 in liquid Luria Bertani (LB) medium alone (for wild-type E. coli BW25113), or LB media supplemented with either 30 μg/ml kanamycin (for selection of the chromosomal mdtM-deletion strain), GSK458 cost or 30 μg/ml kanamycin and 100 μg/ml carbenicillin (Carbenicillin Direct, UK) (for the ΔmdtM BW25113 strain harboring pMdtM or pD22A). Aliquots (4 μl) from a 10-3 to 10-5 logarithmic dilution series of each culture were spotted onto plates layered with LB-agar (1% w/v tryptone, 0.5% w/v yeast extract,

1% w/v NaCl and 1.5% w/v agar). For assays performed with pMdtM and pD22A transformants the LB-agar was supplemented with the appropriate antibiotics and L-arabinose Methamphetamine was added to a final concentration of 0.002% (w/v) to induce expression of the recombinant protein. For all the plate assays, pH of the medium was buffered by 70 mM 1,3-bis[tris(hydroxymethyl)-methylamino] propane (BTP) and pH was adjusted by HCl. Plates were incubated for 24 h at 37°C prior to imaging. Bacterial growth assays in liquid medium A swab of colonies from overnight LB agar plates was used to inoculate 2 ml of LB broth containing the appropriate antibiotic(s) and, where appropriate, 0.002% (w/v) L-arabinose, and grown for 2 h with shaking at 37°C. Cultures were then diluted to an OD600 of 0.02 into 50 ml of fresh LB medium containing the appropriate antibiotic(s) and L-arabinose (0.002% w/v). Media were buffered by 70 mM BTP and pH was adjusted with HCl. Cells were then grown aerobically at 37°C with shaking and the OD600 measured every hour for 15 hours. Assays designed to test the effects of Na+ or K+ ions at alkaline pH on the growth of BW25113 ΔmdtM cells transformed with pMdtM were performed in salt-free LB medium (1% w/v tryptone, 0.5% w/v yeast extract) buffered to the indicated pH with 70 mM BTP.