Table 1.
Genotypes and MICs of the strains carrying the wild type PBPs used.
Table 2.
MICs of Penicillin-G and identified mutations for transformants with increased resistance.
Table 3.
Relative MIC fold change of resistant transformants obtained from the FtsI-based libraries and the DacC-based library against five different β-lactam antibiotics.
Table 4.
MICs of pencillin G for the reconstructed single mutants.
Figure 1.
Sequence alignments of S. enterica FtsI (A) and DacC (B) with PBPs with known structure.
Secondary structure is indicated above the alignment. Strictly conserved residues are shown in white on red background, residues with conserved physico-chemical properties are shown in red on white background. Mutation sites in this study are marked with asterisks below the alignment. The figure was prepared using ESPRIPT [34].
Figure 2.
Domain locations of identified amino acid substitutions in the FtsI (PBP3) and DacC (PBP6) variants that conferred resistance against penicillin G.
(A) Domain locations of mutations in PBP3. (B) Domain locations of mutations in PBP6. The three conserved sites of the PB module are shown. Mutations that, when present singly, confer resistance are indicated in bold.
Figure 3.
Location of mutation sites in known structures.
A. Structure of PBP3 from P. aeruginosa (in complex with carbenicillin, pdb entry 3OCL (27)) The N-terminal n-PB domain is shown in pink and the C-terminal PB domain in pale blue. Residues are named and numbered according to the S. typhimurium FtsI sequence, for non-conserved residues the equivalent residue in P. aeruginosa is shown within parentheses. Carbenicillin (magenta), the catalytic motifs 307STVK310, 359SSN361 and 494KS/TG496 (dark blue), residues equivalent to mutation sites in S. typhimurium DacC (gold, numbered) and residues V344 and Y541 are shown in spacefilling representation. B. P298 (yellow) creates a kink in helix α11 (cartoon and sidechains shown) that seems critical for interactions with the surrounding PB domain (surface representation). The STVK motif is shown in dark blue. C. Structure of PBP6 from E. coli (acyl-enzyme-intermediate with ampicillin, pdb entry [29]). The N-terminal PB domain is shown in pale blue and the C-terminal n-PB domain in pink. Residues are named and numbered according to the S. typhimurium DacC sequence, for non-conserved residues the equivalent residue in E. coli is shown within parentheses. Ampicillin (magenta), the catalytic motifs 66SLTK69, 132SGN134 and 235KTG237 (dark blue) and residues equivalent to mutation sites in S. typhimurium DacC (gold, numbered) are shown in spacefilling representation.