Figure 5. A ReoM T7A exchange affects growth and MurA levels in a ClpC-dependent manner.

(A) Lethality of the reoM T7A and reoM T7D mutations in L. monocytogenes. L. monocytogenes strains EGD-e (wt), LMSW30 (ΔreoM), LMSW57 (ireoM), LMSW52 (ireoM T7A) and LMSW53 (ireoM T7D) were grown in BHI broth ±1 mM IPTG at 37°C. The experiment was repeated three times and average values and standard deviations are shown. (B) Suppression of reoM T7A lethality by deletion of clpC. L. monocytogenes strains EGD-e (wt), LMJR138 (ΔclpC), LMSW52 (ireoM T7A) and LMSW72 (ireoM T7A ΔclpC) were grown in BHI broth ±1 mM IPTG at 37°C. The experiment was repeated three times and average values and standard deviations are shown. (C) Western blot showing cellular levels of MurA (top) and ClpC (middle) in the strains included in panels A and B. For this experiment, strains were grown in BHI broth not containing IPTG at 37°C. IPTG (1 mM) was added to the cultures at an OD₆₀₀ of 0.2 and the cells were collected 2 hr later. Quantification of MurA signals by densitometry is shown below the western blots. Average values and standard deviations calculated from three independent experiments are shown. Asterisks indicate statistically significant differences (p<0.05, t-test). (D) ReoM T7A expression sensitises L. monocytogenes against ceftriaxone. Synergism between ceftriaxone and IPTG in the ireoM T7A strain LMSW52 in a disc diffusion assay with filter discs containing 50 mg/ml ceftriaxone (CRO, left) and 1 mM IPTG (right). For comparison, wild type levels of growth inhibition by ceftriaxone are marked with black circles. Zone of growth inhibition by IPTG in the ireoM T7A mutant is marked with a white circle. Please note that strain LMSW52 shows hetero-resistance against IPTG (two zones of growth inhibition with different resistance levels). Arrows mark the zones of synergism between ceftriaxone and IPTG. (E) Contribution of ReoY and MurZ to the lethal reoM T7A phenotype. L. monocytogenes strains EGD-e (wt), LMSW52 (ireoM T7A), LMSW72 (ireoM T7A ΔclpC), LMSW123 (ireoM T7A ΔreoY) and LMSW124 (ireoM T7A ΔmurZ) were grown in BHI broth containing 1 mM IPTG and growth at 37°C was recorded in a microplate reader. Average values and standard deviations were calculated from an experiment performed in triplicate.