FIG 5.

Increased PBP1b production suppresses the growth defect of ΔrodA ΔponA cells. (A) Diagram of the ponB locus showing the approximate location of the suppressor mutations. The mutations mapped to nucleotide positions 2,959,545, 2,959,217, or 2,959,117 for sup1, sup2, or sup3, respectively, and all were G-to-A transitions. The sup1 strain had one additional mutation in tetA, the sup2 strain had no additional mutations, and the sup3 strain had additional mutations in fkpA, cgtR5, upstream of pitA and gdh, and downstream of phoD. (B) Overnight cultures of the ΔrodA ΔponA strain (JS38) and its sup1, sup2, and sup3 derivatives were grown in BHIS medium at 30°C. Culture densities were normalized to an OD₆₀₀ of 0.5. The normalized cultures were then serially diluted and plated as described in the legend for Fig. 1. Cells were grown on BHI medium at 30°C for 24 h before the plates were photographed. Cells of the strains from panel B (C) or MB001 (WT) and its engineered derivatives containing the sup1 or sup2 mutation in the ponB locus (D) were labeled with Bocillin-FL. Membrane extracts were then prepared from the labeled cells, and 5 μg of total protein for each was subjected to SDS-PAGE. Bocillin-FL-labeled proteins were then detected using a Typhoon 9500 imager with excitation at 488 nm and emission at 530 nm. (E) Overnight cultures of MB001 (WT) with or without a plasmid containing the indicated wild-type or mutant ponB locus fused to a promoterless lacZ reporter were grown in BHIS medium at 30°C. They were diluted 1:200 and grown to an OD₆₀₀ of 0.3 under the same conditions. Cell pellets were then collected and frozen. Thawed cells were then resuspended in Z buffer and permeabilized with toluene and lysozyme. o-Nitrophenyl-β-d-galactopyranoside (ONPG) was added, CaCO₃ was used to stop the reaction, and the absorbance of the reaction measured at 420 nm. Beta-galactosidase activity was determined using the equation activity = [OD₄₂₀ − 1.75 (OD₅₅₀)]/[time (min) × 1 ml vol × OD₆₀₀ × 1,000]. (F) MB001 (WT) and its ΔrodA ΔponA derivative with no plasmid, an empty vector, or a vector expressing mScar-PBP1b (pJWS117) were grown and plated as for panel B. The cells were grown on BHI medium supplemented with theophylline to induce PBP1b production in the pJWS117-containing strain.