Extended Data Fig. 5: Influence of magnesium on accumulation, evaluation of its binding by NMR and influence of magnesium and PMBN on compound accumulation.
From: Porin-independent accumulation in Pseudomonas enables antibiotic discovery
a) Confocal fluorescent images of an intrinsically fluorescent compound, Bisindolylmaleimide X (BIM X) in P. aeruginosa with or without MgCl2. The standard accumulation assay was performed in P. aeruginosa (PAO1) with either DMSO, BIM X (50 μM), or BIM X (50 μM) + MgCl2 (1 mM), until just after the oil removal step. Cells were fixed in 3.7 % formaldehyde in PBS and fluorescence images were taken with a Zeiss 710 multiphoton confocal microscope through a 63x/1.4 oil objective. All samples were excited at 488 nm with an argon laser and fluorescence emission was recorded from 599–689 nm. Images were acquired using Zen Black (Zen 2.3). The images for P. aeruginosa + DMSO and P. aeruginosa + BIM X are the same as in Extended Data Fig. 1. DMSO controls indicate no autofluorescence. Images are representative of n = 3 biologically independent samples. b) Evaluation of accumulation of compounds in E. coli MG1655 upon cotreatment with MgCl2 (1 mM). c) Determination of Mg2+ interactions with polyamines. Ethylenediaminetetraacetic acid (EDTA), a compound known to chelate to Mg2+ in solution, clearly shows a marked chemical shift change in the NMR as well as changes in coupling when comparing EDTA alone to EDTA plus Mg2+. When this same experiment was performed with norspermine, there were no observable changes in coupling or chemical shift in the presence of MgCl2, suggesting a lack of perturbation of any electronic environment on the molecule, thus, a lack of interaction between Mg2+ and norspermine. Compounds were evaluated at a final concentration of 1 mM and MgCl2 was at a final concentration of 20 mM to maintain the same Mg2+-to-compound ratio as in the accumulation experiments. The pD of all solutions was adjusted to 7.2 prior to analysis. d) The same set of compounds as in Extended Data Fig. 5b showed a statistically significant increase in accumulation in P. aeruginosa PAO1 upon cotreatment with the permeabilizer PMBN (8 μg/ml). All structures and accumulation values are listed in Supplementary Table 4b. For samples with additives in Extended Data Fig. 5b & d, compounds 23 and 35 did not meet the mass spec standards and are thus excluded from this analysis. n = 3 biologically independent samples. The average and s.e.m are reported for accumulation values. Statistical significance was determined using a two-sample Welch’s t-test (one-tailed test, assuming unequal variance). Statistically significant accumulation differences for compounds in P. aeruginosa PA14 versus P. aeruginosa PA14 with PMBN treatment or E. coli MG1655 versus E. coli MG1655 with MgCl2 treatment are indicated with asterisks (n.s. not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).
