Fig. 4 Functional confirmation of genes associated with isopropanol tolerance in E. faecium. Functional confirmation of genes associated with isopropanol.

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Fig. 4 Functional confirmation of genes associated with isopropanol tolerance in E. faecium. Functional confirmation of genes associated with isopropanol tolerance in E. faecium. (A) Map of the 197-kb E. faecium plasmid showing the 70-kb region associated with isopropanol tolerance (blue) and the two carbohydrate PTS loci, including the 6.3-kb PTS-2 locus (purple) deleted by allelic exchange in the E. faecium reference strain Ef_aus0233. (B) Layout of the region around Ef_aus0233 chromosome position 519,608 showing the region deleted by allelic exchange in the gene 00501 encoding a putative galactoside symporter. Dark blue arrows indicate position of primers used to generate the recombination substrate for mutant construction. Primer positions: (a) 521,394 to 521,371; (b) 520,396 to 520,420; (c) 518,946 to 518,918; (d) 517,961 to 517,985. (C) Impact of the rpoBH486Y mutation, 197-kb plasmid ΔPTS-2, and Δ00501 galactoside symporter mutation on the ability of Ef_aus0233 to survive exposure to isopropanol. The means and SDs for biological triplicate experiments, with no differences between any of the mutants and wild-type (WT) E. faecium (table S9), are shown. (D) Growth phenotypes of the same rpoBH486Y mutation, 197-kb plasmid ΔPTS-2, and Δ00501 galactoside symporter mutation in the presence of 3% (v/v) isopropanol. Fold-change difference in doubling time for each mutant compared to wild-type E. faecium is shown. The phenotypes of the repaired mutants relative to wild-type E. faecium are also depicted. The null hypothesis (no difference between mean doubling time of mutant and repaired mutant or wild-type E. faecium) was rejected for P < 0.01, unpaired Mann-Whitney test (**P < 0.01, ***P < 0.0001; table S10). Error bars depict SDs. All data points are shown for at least three biological replicates and three technical replicates for each condition. Sacha J. Pidot et al., Sci Transl Med 2018;10:eaar6115 Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works