Magnesium promotes bacterial resistance to antibiotics.

A. MIC of ATCC 33787 and V. parahaemolyticus to BLFX in SWT or LB medium by microtitre-dilution- method. B. MIC of ATCC 33787 to BLFX in SWT with the replacement of MgCl2 with MgSO4 MgCl2 by microtitre-dilution-method. C. MIC of ATCC 33787 to BLFX in SWT with the indicated concentrations of MgCl2 by microtitre-dilution-method. D. MIC of ATCC 33787 to BLFX in the indicated concentrations of BLFX and MgCl2 by Oxford cup test. E. MIC of ATCC 33787 to other quinolones in SWT with the indicated concentrations of MgCl2 by microtitre-dilution-method. F. MIC of ATCC 33787 to other classes of antibiotics in SWT with the indicated concentrations of MgCl2 by microtitre-dilution-method.

Mg2+ elevates intracellular Mg2+ and promotes balofloxacin uptake.

A. Oxford cup test of 0, 12.5, 25, 50, 100, 200 μg/mL BLFX to ATCC33787 following by separately incubation with the indicated concentrations of MgCl2 for 5 h. B. Inhibitory curve of data (A). C. Binding efficacy of (A). D. Intracellular BLFX of ATCC 33787 in SWT with the indicated concentrations of MgCl2 and 60 μg/mL BLFX. E. Intracellular Mg2+ of ATCC 33787 in SWT with the indicated concentrations of MgCl2. F. Western blot for abundance of TolC in the presence of MgCl2. G. LPS quantification at indicated concentrations of MgCl2. H. MIC of ATCC 33787 and its mutants ΔwaaF ΔlpxA ΔlpxC in SWT with the indicated concentrations of MgCl2, which is measured by microtitre-dilution-method.

Mg2+-induced metabolomics.

A. Differential metabolomes in the absence or presence of the indicated concentrations of MgCl2. Yellow color and blue color indicate increase and decrease of metabolites relative to the median metabolite level, respectively (see color scale). B. PCA analysis of different concentrations of MgCl2-induced metabolomes. Each dot represents the technical replicate of samples in the plot. C. S-plot generated from OPLS-DA. Predictive component p [1] and correlation p(corr) [1] differentiate 0, 0.78, 3.125 mM MgCl2 from 12.5, 50, 200 mM MgCl2. Predictive component p[2] and correlation p(corr)[2] separate 0, 0.78, 50, 200 mM MgCl2 from 3.125, 12.5 mM MgCl2. The triangle represents metabolites in which candidate biomarkers are marked. D. Enriched pathways by differential abundances of metabolites. E. Scatter plot of palmitic acid and stearic acid in the indicated concentrations of MgCl2, which comes from data (A). F. Synergy analysis for alofloxacin with palmitic acid for V. algilyticus. Synergy is represented using a color scale or an isobologram, which compares the dose needed for 50% inhibition for synergistic agents (white) and non-synergistic (i.e., additive) agents (purple).

Mg2+ promotes biosynthesis of fatty acids.

A. qRT-PCR for expression of fatty acid biosynthesis genes in the absence or presence of MgCl2. B. Western blot for abundance of proteins responsible for conversion to unsatisfied fatty acid biosynthesis or satisfied fatty acid in the absence or presence of MgCl2. C. qRT-PCR for expression of genes encoding conversion from unsatisfied fatty acid biosynthesis to satisfied fatty acid in the absence or presence of MgCl2. D. Diagram for biosynthesis of saturated and unsaturated acids. E. Synergy analysis for balofloxacin with triclosan + oxazole-2- amine for ATCC33787. Synergy is represented using a color scale or an isobologram, which compares the dose needed for 50% inhibition for synergistic agents (blue) and non-synergistic (i.e., additive) agents (red). F. Diagram for degradation of fatty acids. G. qRT-PCR for expression of genes encoding fatty acid degradation in the absence or presence of the indicated concentrations of MgCl2. H. Western blot for abundance of FadL in the absence or presence of the indicated concentrations of MgCl2. I. qRT-PCR for expression of regulatory genes of fatty acid biosynthesis in the absence or presence of the indicated concentrations of MgCl2. J. Western blot for abundance of FadR in the absence or presence of the indicated concentrations of MgCl2. K and L. Synergy analysis for MgCl2 with BLFX for ΔfadR (L) and ΔarcA (M). Synergy is represented using a color scale or an isobologram, which compares the dose needed for 50% inhibition for synergistic agents (while) and non-synergistic (i.e., additive) agents (red). M , N and O qRT-PCR for expression of genes encoding biosynthesis of fatty acids and degradation of fatty acids (M) in ATCC33787 and ΔfadR (N) and ΔarcA (O)in the presence or absence of 200 mM MgCl2.

LC-MS targeted 16-carbon and 18-carbon fatty acids and role of palmitic acids and linolenic acids in BLFX resistance.

A. Scatter plots of 16-carbon and 18-carbon fatty acids, detected by LC-MS. B. Scatter plots for total saturated fatty acid and unsaturated fatty acid with16-carbon and 18-carbon in data (A). C. Synergy analysis for BLFX with linolenic acid for ATCC 33787. Synergy is represented using a color scale or an isobologram, which compares the dose needed for 50% inhibition for synergistic agents (while) and non-synergistic (i.e., additive) agents (purple). D. LC- MS for abundance of intracellular linolenic acid and palmitic acid of ATCC 33787 in synergy of the indicated exogenous linolenic acid and palmitic acid. E. Synergy analysis for linolenic acid with palmitic acid for BLFX-mediated killing to ATCC 33787. Synergy is represented using a color scale or an isobologram, which compares the dose needed for 50% inhibition for synergistic agents (blue) and non-synergistic (i.e., additive) agents (red). F. Bliss analysis in data (E).

Effect of Mg2+ on phospholipid biosynthesis.

A. Heatmap showing differential abundance of lipid. B. Abundance of ATCC33787 phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) in the indicated concentrations of MgCl2. C. Scatter plots of PG and PE with different saturation in the presence of the indicated MgCl2. D. PCA analysis of different concentrations of MgCl2-induced phosphoglyceride metabolomes. Each dot represents the technical replicate of samples in the plot. E. S-plot generated from OPLS-DA. Predictive component p [1] and correlation p (corr) [1] differentiate 0 and 3.125 mM MgCl2 from 50 and 200 mM MgCl2. F. Scatter plot of biomarkers in data (E). G. Diagram showing phosphoglyceride metabolism. H. qRT-PCR for expression of genes encoding phosphoglyceride metabolism in the absence or the indicated concentrations of MgCl2. I. Activity of PGS and PSS in the absence or presence of the indicated concentrations of MgCl2. J. Activity of recombinant PSS in the absence or presence of the indicated concentrations of MgCl2. K and L Synergy analysis for MgCl2 with BLFX for ΔplsB (K) and ΔpgpA (L). Synergy is represented using a color scale or an isobologram, which compares the dose needed for 50% inhibition for synergistic agents (blue) and non-synergistic (i.e., additive) agents (red).

Mg2+ regulates membrane polarization, permeability and fluidity to confer balofloxacin resistance.

A. and B. Depolarization (A) and PMF (B) of ATCC33787 in the absence or indicated concentrations of MgCl2. C and D. Dynamic depolarization (D) Membrane fluidity of ATCC33787 in the absence or presence of the indicated concentrations of MgCl2, detected in fluorescence microscopy. E. Membrane permeability of ATCC33787 in the absence or presence of the indicated concentrations of MgCl2. F and G. Membrane permeability of ATCC33787 cultured in palmitic acid (F) or linolenic acid (G) in the indicated concentrations of MgCl2. H and I. Membrane permeability of ΔfadR (H) and ΔarcA (I) in the absence or presence of the indicated concentrations of MgCl2. J. Membrane permeability of ΔpgpA in the absence or presence of the indicated concentrations of MgCl2. K. Intracellular BLFX of ATCC33787 in the presence of palmitic acid, linolenic acid, PG, and PE (lefe panel) or in ΔfadR and ΔpgpA mutants (right panel). L. Diagram for mechanisms by which Mg2+- mediated resistance to BLFX.

MIC of ATCC33787 to BLFX in the absence or presence of the indicated concentrations of KCl or CaCl2.

Metabolic profiles of V. alginolyticus in different concentrations of MgCl2.

A. Reproducibility

B. Percentage of metabolites in every category

C. Heatmap of metabolites

Heatmap and Z score plots of differential metabolites.

A. Heatmap of differential metabolites

B-F. Z score plots of differential metabolites.

Pathway enrichment of differential metabolites.

A. Pathway enrichment of differential metabolites

B. Differential metabolites in enriched pathways

Scatter plots of differential metabolites identified by S-plot.

Lipidomes in the different concentrations of MgCl2.

A. Area of fatty acids in the presence of indicated concentrations of MgCl2.

B. Percentage of lipids, saturated fatty acid and unsaturated fatty acid in the presence of indicated concentration of MgCl2.

C. Volcano plots of lipidomics of indicated concentration of MgCl2 as compared to non-treated control.

D. Relative abundance of saturated fatty acids, unsaturated fatty acids and lipids in the presence of indicated concentrations of MgCl2.

E. Relative percentage of indicated lipids.

Comparison in components in LBS and SWT

Primes used in the present study

Primers used in the present study for construction of gene-deleted mutants