Fig. 4

Impact of cellular localization and protein features of the MBLs on fitness cost. a Schematic representation of the chimeric MBLs variants with altered cellular localizations and engineered signal peptides. NT: N-terminus and CT: C-terminus. OM: protein lipidated and anchored to outer membrane. S: soluble periplasmic protein. C: cysteine anchoring residue into lipobox sequence. A: alanine residue. By mutation of the cysteine residue by an alanine residue, C26A, the lipidation site is removed resulting in a soluble variant. The same color coding is used for each variant throughout this Figure. b Growth curves of E.coli strains expressing bla_NDM-1 or bla_VIM-2 with altered cellular localizations in LB at 37 °C with 50 µM of IPTG (top and medium panels). The growth was monitored as above. Data represent the mean value (±s.d) of three independent experiments. Bottom panel: Protein levels of NDM-1, VIM-2 and their chimeras with altered cellular localizations by immunoblotting in whole cells from E. coli expressing each MBL after induction at 20 µM with IPTG. Red arrows indicate the precursor forms. c Growth curves of E. coli strains expressing bla_IMP-1, bla_VIM-2, and their chimeras with their exchanged signal peptidesI1-VIM-2 and V2-IMP-1 (top panel). Growth curves of E. coli strains expressing bla_VIM-1, bla_VIM-2, and their chimeras with their exchanged signal peptides (medium panel). Bottom panel shows the protein levels of VIM-2, VIM-1, and their chimeras with their signal peptides swapped, by immunoblot analysis. Lower panels show loading control (GroEL). d Bacterial viability in a spot plating assay. Overnight cultures from E. coli expressing VIM-2, IMP-1, VIM-1, and their chimeras were serially diluted and 10 µl aliquots spotted onto LB agar without (−) or with the addition of 50 µM of IPTG (+). Colonies were allowed to develop for 24 h at 37 °C. Panels c (bottom panel) and d are representative of two independent experiments. Source data are provided as a Source Data file