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. 2016 Nov 18;84(12):3597–3607. doi: 10.1128/IAI.00647-16

FIG 2.

FIG 2

L. monocytogenes encodes two functional enoyl-acyl carrier protein reductase isoforms. (A) Growth of the temperature-sensitive E. coli strain JP1111 [fabI(Ts)] transformed with the empty plasmid (Control) or plasmids expressing either S. aureus FabI (SaFabI), predicted L. monocytogenes FabK1 (LmFabK1; LMO0814), predicted L. monocytogenes FabI (LmFabI; LMO0970), predicted L. monocytogenes FabL (LmFabL; LMO1688), or predicted L. monocytogenes FabK2 (LmFabK2; LMO2170) under the permissive growth condition (30°C). (B) Growth of the same strains at the nonpermissive temperature (42°C). (C) The expression of the predicted L. monocytogenes enoyl-acyl carrier protein reductases in JP1111 was verified via Western blot analysis. JP1111 cells expressing the enoyl-acyl carrier protein reductases were grown at 30°C to an A600 of 1. The cells were lysed, and the supernatant was separated from the cell debris. Western blot analysis against the supernatant fraction using an antihistidine tag was performed to ensure that the predicted enoyl-acyl carrier protein reductase was expressed. All four predicted enoyl-acyl carrier protein reductases (LmFabK1, 32.4 kDa; LmFabI, 28.3 kDa; LmFabL, 27.2 kDa; LmFabK2, 32.5 kDa; each with 2.1 kDa added from the N-terminal histidine tag from pPJ131 plasmid) were expressed in JP1111. Data shown as a representative blot are from 2 biological replicates. (D) MIC of AFN-1252 against the E. coli strain ANS1 (ΔtolC) expressing empty plasmid (control), S. aureus FabI (SaFabI), L. monocytogenes FabK (LmFabK1; LMO0814), L. monocytogenes FabI (LmFabI; LMO0970), and L. monocytogenes FabL (LmFabL; LMO1688).